No full-text available
To read the full-text of this research,
you can request a copy directly from the author.
Directed Evolution of Nucleic Acid Enzymes
Abstract
Just as Darwinian evolution in nature has led to the development of many sophisticated enzymes, Darwinian evolution in vitro has proven to be a powerful approach for obtaining similar results in the laboratory. This review focuses on the development of nucleic acid enzymes starting from a population of random-sequence RNA or DNA molecules. In order to illustrate the principles and practice of in vitro evolution, two especially well-studied categories of catalytic nucleic acid are considered: RNA enzymes that catalyze the template-directed ligation of RNA and DNA enzymes that catalyze the cleavage of RNA. The former reaction, which involves attack of a 2'- or 3'-hydroxyl on the alpha-phosphate of a 5'-triphosphate, is more difficult. It requires a comparatively larger catalytic motif, containing more nucleotides than can be sampled exhaustively within a starting population of random-sequence RNAs. The latter reaction involves deprotonation of the 2'-hydroxyl adjacent to the cleavage site, resulting in cleaved products that bear a 2',3'-cyclic phosphate and 5'-hydroxyl. The difficulty of this reaction, and therefore the complexity of the corresponding DNA enzyme, depends on whether a catalytic cofactor, such as a divalent metal cation or small molecule, is present in the reaction mixture.
... 314 It requires the ribozyme to ligate a chimeric DNA-RNA substrate that has the sequence of the promoter element for a DNA-dependent RNA polymerase. 314,315 After reverse transcription, cDNAs derived from an active ribozyme containing a functional promoter element will be generated. Multiple copies of RNA per copy of DNA template can be obtained after repeated cycles with reverse transcriptase and RNA polymerase. ...
... Multiple copies of RNA per copy of DNA template can be obtained after repeated cycles with reverse transcriptase and RNA polymerase. 314,315 Population size, sequence diversity, and selection pressure can all be controlled. The rapid pace of continuous in vitro evolution makes it powerful and allows one to perform tens to hundreds of generations per day. ...
... The rapid pace of continuous in vitro evolution makes it powerful and allows one to perform tens to hundreds of generations per day. 314,315 In addition, since the workflow of this method is easy to handle, only minimal human intervention is required. However, it has limitations because several criteria must be met for ribozyme-catalyzed reactions to occur. ...
... But this apparent paradox has a possible explanation. Ribozymes with ligase activity (a single condensation reaction with two oligonucleotide substrates) isolated in vitro can also catalyze the template-directed addition of only three successive nucleotides (Ekland and Bartel 1996;Joyce et al. 2004). That is, one ribozyme can possess the ability for both triplet-and mononucleotide-based polymerization. ...
... The basic chemistry needed for template-directed polymerization-in which the 3'-OH in a nucleotide or oligonucleotide5triphosphate group phosphoanhydride bond is attacked-occurs within the group I intron of a natural ribozyme (Vincens and Cech 2009). The chemistry of polymerization has been discussed in a lot of detail in the academic literature, but one the most notable points is that dual metal-ion chemistry is leveraged for this reaction in both extant protein-based RNA and DNA polymerases and in vitro selected ribozyme RNA polymerases and ligases (Joyce 2004;Stahley and Strobel 2005;Strobel and Cochrane 2007). ...
The origin of genetic systems is the central problem in the study of the origin of life for which various explanatory hypotheses have been presented. One model suggests that both ancestral transfer ribonucleic acid (tRNA) molecules and primitive ribosomes were originally involved in RNA replication (Campbell 1991). According to this model the early tRNA molecules catalyzed their own self-loading with a trinucleotide complementary to their anticodon triplet, while the primordial ribosome (protoribosome) catalyzed the transfer of these terminal trinucleotides from one tRNA to another tRNA harboring the growing RNA polymer at the 3´-end.
Here we present the notion that the anticodon-codon-like pairs presumably located in the acceptor stem of primordial tRNAs (Rodin et al. 1996) (thus being and remaining, after the code and translation origins, the major contributor to the RNA operational code (Schimmel et al. 1993)) might have originally been used for RNA replication rather than translation; these anticodon and acceptor stem triplets would have been involved in accurately loading the 3’-end of tRNAs with a trinucleotide complementary to their anticodon triplet, thus allowing the accurate repair of tRNAs for their use by the protoribosome during RNA replication.
We propose that tRNAs could have catalyzed their own trinucleotide self-loading by forming catalytic tRNA dimers which would have had polymerase activity. Therefore, the loading mechanism and its evolution may have been a basic step in the emergence of new genetic mechanisms such as genetic translation. The evolutionary implications of this proposed loading mechanism are also discussed.
... The development and application of methods of artificial evolution such as SELEX and in vitro selection has led to the discovery of myriad RNA and DNA molecules with interesting and useful properties [1][2][3][4][5]. For instance, aptamers have been identified that bind diverse classes of ligands, often with affinities in the nanomolar to picomolar range [6]. ...
... DNA and RNA motifs with a range of functions have been identified in artificial evolution experiments [1][2][3][4][5]. In most cases these motifs are initially isolated from random sequence libraries containing~10 15 different sequences. ...
Methods of artificial evolution such as SELEX and in vitro selection have made it possible to isolate RNA and DNA motifs with a wide range of functions from large random sequence libraries. Once the primary sequence of a functional motif is known, the sequence space around it can be comprehensively explored using a combination of random mutagenesis and selection. However, methods to explore the sequence space of a secondary structure are not as well characterized. Here we address this question by describing a method to construct libraries in a single synthesis which are enriched for sequences with the potential to form a specific secondary structure, such as that of an aptamer, ribozyme, or deoxyribozyme. Although interactions such as base pairs cannot be encoded in a library using conventional DNA synthesizers, it is possible to modulate the probability that two positions will have the potential to pair by biasing the nucleotide composition at these positions. Here we show how to maximize this probability for each of the possible ways to encode a pair (in this study defined as A-U or U-A or C-G or G-C or G.U or U.G). We then use these optimized coding schemes to calculate the number of different variants of model stems and secondary structures expected to occur in a library for a series of structures in which the number of pairs and the extent of conservation of unpaired positions is systematically varied. Our calculations reveal a tradeoff between maximizing the probability of forming a pair and maximizing the number of possible variants of a desired secondary structure that can occur in the library. They also indicate that the optimal coding strategy for a library depends on the complexity of the motif being characterized. Because this approach provides a simple way to generate libraries enriched for sequences with the potential to form a specific secondary structure, we anticipate that it should be useful for the optimization and structural characterization of functional nucleic acid motifs.
... In addition, the cooperative stabilization of duplex nucleic acids by metal-ion bridging of base mismatches, for example, T-Hg 2+ -T or C-Ag + -C, and their separation by ion binding ligands, such as cysteine [8][9][10] (e) demonstrate further the capabilities to control the structures and stabilities of nucleic acid assemblies. Functional information embedded in the nucleic acid biopolymers includes sequence-specific recognition and binding of molecular or macromolecular ligands (aptamers) [11][12][13] (f), and sequence-controlled catalytic properties of nucleic acids, characteristic of DNAzymes 14,15 or nucleoapzymes 16 , (g). Besides, a variety of enzymes control the structures of nucleic acids. ...
... The two strands were deposited on the graphene surface, where quenching of the luminescent AgNCs, associated with the two probes occurred. Subjecting the graphene oxide scaffold, on which the AgNCs-modified probes (15) and (16) were deposited, to the HBV gene (18) stimulated the removal of the AgNCs-modified probe (15) from the graphene surface, leading to the switch-on luminescence of the released NCs associated with the (15)/HBV gene duplex. Similarly, the interaction of the graphene oxide modified with the two types of AgNCs/DNA probes with the HIV gene (17) led to the release of the AgNCs-functionalized (16)/HIV gene duplex and to the switched-on luminescence of the 775 nm luminescent AgNCs. ...
Since the discovery of the double-helix structure in 1953, nucleic acids have been developed from natural genetic codes into functional building blocks in a wide range of biotechnology and materials sciences. Taking advantage of their design diversity and biocompatibility, functional nucleic acids facilitate the "bottom-up" fabrication of nanomaterials that are highly potential for molecular medicine to treat different diseases, such as cancers. The present perspective article introduces recent advances in the use of these unique properties of nucleic acid biopolymers for biomedical applications. Specifically, nanomaterial/ nucleic acid hybrid structures for sensing, controlled drug release, programmable intracellular imaging, and apoptosis, as well as logic calculation, are discussed. Furthermore, the detailed operation for both extracellular and intracellular bioactivity regulation with these new design functional nucleic acid nanostructures are fully illustrated.
... DNA enzymes, also known as DNAzymes, are DNA sequences able to catalyze chemical reactions in a similar manner as protein enzymes. However, contrary to ribozymes or protein enzymes, DNAzymes cannot be found in nature and are generated through an in vitro selection process [4]. Since their discovery, many DNAzymes have been selected, exhibiting a wide variety of functionalities, such as redox [5], ligation [6], phosphorylation [7] or RNA cleavage [8]. ...
DNA-based enzymes, or DNAzymes, are single-stranded DNA sequences with the ability to catalyze various chemical reactions, including the cleavage of the bond between two RNA nucleotides. Lately, an increasing interest has been observed in these RNA-cleaving DNAzymes in the biosensing and therapeutic fields for signal generation and the modulation of gene expression, respectively. Additionally, multiple efforts have been made to study the effects of the reaction environment and the sequence of the catalytic core on the conversion of the substrate into product. However, most of these studies have only reported alterations of the general reaction course, but only a few have focused on how each individual reaction step is affected. In this work, we present for the first time a mathematical model that describes and predicts the reaction of the 10–23 RNA-cleaving DNAzyme. Furthermore, the model has been employed to study the effect of temperature, magnesium cations and shorter substrate-binding arms of the DNAzyme on the different kinetic rate constants, broadening the range of conditions in which the model can be exploited. In conclusion, this work depicts the prospects of such mathematical models to study and anticipate the course of a reaction given a particular environment.
... Deoxyribozymes, also known as DNAzymes, DNA enzymes, or catalytic DNA, have been established as biological catalysts acting similarly to other protein enzymes and ribozymes [1]. Their sequences are selected through in vitro selection methods [2][3][4][5]. Because of their stability in physiological conditions, high sensitivity, and selectivity, DNAzymes are widely applied in practices [6], such as heavy metal ion detectors [7,8], biosensors [9][10][11], cellular imaging [12][13][14] and diagnostics [15,16]. ...
DNAzyme is a class of DNA molecules that can perform catalytic functions with high selectivity towards specific metal ions. Due to its potential applications for biosensors and medical therapeutics, DNAzyme has been extensively studied to characterize the relationships between its biochemical properties and functions. Similar to protein enzymes and ribozymes, DNAzymes have been found to undergo conformational changes in a metal–ion–dependent manner for catalysis. Despite the important role the conformation plays in the catalysis process, such structural and dynamic information might not be revealed by conventional approaches. Here, by using the single–molecule fluorescence resonance energy transfer (smFRET) technique, we were able to investigate the detailed conformational dynamics of a uranyl–specific DNAzyme 39E. We observed conformation switches of 39E to a folded state with the addition of Mg2+ and to an extended state with the addition of UO22+. Furthermore, 39E can switch to a more compact configuration with or without divalent metal ions. Our findings reveal that 39E can undergo conformational changes spontaneously between different configurations.
... They also indicate that nucleic acids can catalyze a wide range of simple reactions with considerable rate enhancements. Details of many of these studies and insights from pioneers of the field have been described in a number of excellent reviews [39][40][41][42][43][44]. These focus on topics such as the catalytic diversity of nucleic acids, the types of ligands that can be recognized by aptamers, and the structural basis of nucleic acid function. ...
For many decades it was thought that information storage and information transfer were the main functions of nucleic acids. However, artificial evolution experiments have shown that the functional potential of DNA and RNA is much greater. Here I provide an overview of this technique and highlight recent advances which have increased its potency. I also describe how artificial evolution has been used to identify nucleic acids with extreme functions. These include deoxyribozymes that generate unusual products such as light, tiny motifs made up of fewer than ten nucleotides, ribozymes that catalyze complex reactions such as RNA polymerization, information‐rich sequences that encode overlapping ribozymes, motifs that catalyze reactions at rates too fast to be followed by manual pipetting, and functional nucleic acids which are active in extreme conditions. Such motifs highlight the limits of our knowledge and provide clues about as of yet undiscovered functions of DNA and RNA.
... The smallest ribozymes known today are 30 to 100 nucleotides long [22][23][24]. More complex ribozymes that could, e.g., assist replication are likely to have a minimum length of more than 150 nucleotides [25][26][27]. For polymers of a length between 30 to 150, a total of 10 18 to 10 90 distinct sequences are possible. ...
The RNA world is one of the principal hypotheses to explain the emergence of living systems on the prebiotic Earth. It posits that RNA oligonucleotides acted as both carriers of information as well as catalytic molecules, promoting their own replication. However, it does not explain the origin of the catalytic RNA molecules. How could the transition from a pre-RNA to an RNA world occur? A starting point to answer this question is to analyze the dynamics in sequence space on the lowest level, where mononucleotide and short oligonucleotides come together and collectively evolve into larger molecules. To this end, we study the sequence-dependent self-assembly of polymers from a random initial pool of short building blocks via templated ligation. Templated ligation requires two strands that are hybridized adjacently on a third strand. The thermodynamic stability of such a configuration crucially depends on the sequence context and, therefore, significantly influences the ligation probability. However, the sequence context also has a kinetic effect, since non-complementary nucleotide pairs in the vicinity of the ligation site stall the ligation reaction. These sequence-dependent thermodynamic and kinetic effects are explicitly included in our stochastic model. Using this model, we investigate the system-level dynamics inside a non-equilibrium `RNA reactor’ enabling a fast chemical activation of the termini of interacting oligomers. Moreover, the RNA reactor subjects the oligomer pool to periodic temperature changes inducing the reshuffling of the system. The binding stability of strands typically grows with the number of complementary nucleotides forming the hybridization site. While shorter strands unbind spontaneously during the cold phase, larger complexes only disassemble during the temperature peaks. Inside the RNA reactor, strand growth is balanced by cleavage via hydrolysis, such that the oligomer pool eventually reaches a non-equilibrium stationary state characterized by its length and sequence distribution. How do motif-dependent energy and stalling parameters affect the sequence composition of the pool of long strands? As a critical factor for self-enhancing sequence selection, we identify kinetic stalling due to non-complementary base pairs at the ligation site. Kinetic stalling enables cascades of self-amplification that result in a strong reduction of occupied states in sequence space. Moreover, we discuss the significance of the symmetry breaking for the transition from a pre-RNA to an RNA world.
... Meanwhile, sequence-specific nucleic acids (aptamers) possess selective recognition properties towards low-molecular-weight or macromolecular ligands yielding high-affinity supramolecular complexes. [97][98][99] In addition, sequence-dictated catalytic properties of nucleic acids (DNAzymes), such as the hemin/G-quadruplex peroxidase-mimicking DNAzyme [100,101] or metal-ion cofactor sequence-specific DNAzymes, [102][103][104][105][106] have attracted growing interest in recent years. The unique catalytic and binding functions of nucleic acids were applied to develop supramolecular hybrid systems mimicking native enzymes. ...
Mimicking photosynthesis using artificial systems, as a means for solar energy conversion and green fuel generation, is one of the holy grails of modern science. This perspective presents recent advances towards developing artificial photosynthetic systems. In one approach, native photosystems are interfaced with electrodes to yield photobioelectrochemical cells that transform light energy into electrical power. This is exemplified by interfacing photosystem I (PSI) and photosystem II (PSII) as an electrically contacted assembly mimicking the native Z‐scheme, and by the assembly of an electrically wired PSI/glucose oxidase biocatalytic conjugate on an electrode support. Illumination of the functionalized electrodes led to light‐induced generation of electrical power, or to the generation of photocurrents using glucose as the fuel. The second approach introduces supramolecular photosensitizer nucleic acid/electron acceptor complexes as functional modules for effective photoinduced electron transfer stimulating the subsequent biocatalyzed generation of NADPH or the Pt‐nanoparticle‐catalyzed evolution of molecular hydrogen. Application of the DNA machineries for scaling‐up the photosystems is demonstrated. A third approach presents the integration of artificial photosynthetic modules into dynamic nucleic acid networks undergoing reversible reconfiguration or dissipative transient operation in the presence of auxiliary triggers. Control over photoinduced electron transfer reactions and photosynthetic transformations by means of the dynamic networks is demonstrated.
... This led to the development of methods of artificial evolution which made it possible to isolate DNA and RNA molecules with desired functional properties from large random sequence pools (1)(2)(3). Application of these methods revealed that both DNA and RNA molecules can bind ligands with high affinity and specificity and catalyze a wide range of reactions (4)(5)(6)(7). An advantage of constructing functional motifs from nucleic acids rather than proteins is that they are typically less expensive to synthesize and simpler to prepare (8). ...
Artificial evolution experiments typically use libraries of ∼1015 sequences and require multiple rounds of selection to identify rare variants with a desired activity. Based on the simple structures of some aptamers and nucleic acid enzymes, we hypothesized that functional motifs could be isolated from significantly smaller libraries in a single round of selection followed by high-throughput sequencing. To test this idea, we investigated the catalytic potential of DNA architectures in which twelve or fifteen randomized positions were embedded in a scaffold present in all library members. After incubating in either the presence or absence of lead (which promotes the nonenzymatic cleavage of RNA), library members that cleaved themselves at an RNA linkage were purified by PAGE and characterized by high-throughput sequencing. These selections yielded deoxyribozymes with activities 8- to 30-fold lower than those previously isolated under similar conditions from libraries containing 1014 different sequences, indicating that the disadvantage of using a less diverse pool can be surprisingly small. It was also possible to elucidate the sequence requirements and secondary structures of deoxyribozymes without performing additional experiments. Due to its relative simplicity, we anticipate that this approach will accelerate the discovery of new catalytic DNA and RNA motifs.
... DNAzymes are artificial single stranded DNA oligonucleotides that are selected in vitro by directed evolution and can catalyze specific reactions, e. g. nucleic acid cleavage or ligation. [72] The first reported DNAzyme possessing an RNA ligase activity was discovered by the working group of Scott Silverman in 2003. [73] However, in an Mg 2 + mediated manner, unnatural branched 2'-5' phosphodiester linkages were formed between a 2',3'-cyclic phosphate and a 5'-hydroxyl group of two RNA oligonucleotides by this DNAzyme. ...
The introduction of chemical modifications into long RNA molecules at specific positions for visualization, biophysical investigations, diagnostic and therapeutic applications still remains challenging. In this review, we present recent approaches for covalent internal labeling of long RNAs. Topics included are the assembly of large modified RNAs via enzymatic ligation of short synthetic oligonucleotides and synthetic biology approaches preparing site‐specifically modified RNAs via in vitro transcription using an expanded genetic alphabet. Moreover, recent approaches to employ deoxyribozymes (DNAzymes) and ribozymes for RNA labeling and RNA methyltransferase based labeling strategies are presented. We discuss the potentials and limits of the individual methods, their applicability for RNAs with several hundred to thousands of nucleotides in length and indicate future directions in the field.
... DNA oligonucleotides can also have functional properties, such as in molecular recognition and catalysis, the latter known as DNAzymes [76]. Many peroxidase mimics are DNA-based catalysts and oxidize common substrates like TMB and ABTS in the presence of H 2 O 2 , which is of interest because colorimetric products enable the development of optical sensors [77]. ...
As part of the biomimetic enzyme field, nanomaterial-based artificial enzymes, or nanozymes, have been recognized as highly stable and low-cost alternatives to their natural counterparts. The discovery of enzyme-like activities in nanomaterials triggered a broad range of designs with various composition, size, and shape. An overview of the properties of nanozymes is given, including some examples of enzyme mimics for multiple biosensing approaches. The limitations of nanozymes regarding lack of selectivity and low catalytic efficiency may be surpassed by their easy surface modification, and it is possible to tune specific properties. From this perspective, molecularly imprinted polymers have been successfully combined with nanozymes as biomimetic receptors conferring selectivity and improving catalytic performance. Compelling works on constructing imprinted polymer layers on nanozymes to achieve enhanced catalytic efficiency and selective recognition, requisites for broad implementation in biosensing devices, are reviewed. Multimodal biomimetic enzyme-like biosensing platforms can offer additional advantages concerning responsiveness to different microenvironments and external stimuli. Ultimately, progress in biomimetic imprinted nanozymes may open new horizons in a wide range of biosensing applications.
... These parts can then be templated as substrate fragments (food set) for the catalytic production of a new copy of the ribozyme (Figure 7a). By manipulating the base-pairing recognition while keeping the catalytic structural element the same, cross-catalytic reproduction (instead of self-reproduction) of two ribozymes is achieved (Figure 7a) [156,157]. ...
Understanding the emergence of life from (primitive) abiotic components has arguably been one of the deepest and yet one of the most elusive scientific questions. Notwithstanding the lack of a clear definition for a living system, it is widely argued that heredity (involving self-reproduction) along with compartmentalization and metabolism are key features that contrast living systems from their non-living counterparts. A minimal living system may be viewed as “a self-sustaining chemical system capable of Darwinian evolution”. It has been proposed that autocatalytic sets of chemical reactions (ACSs) could serve as a mechanism to establish chemical compositional identity, heritable self-reproduction, and evolution in a minimal chemical system. Following years of theoretical work, autocatalytic chemical systems have been constructed experimentally using a wide variety of substrates, and most studies, thus far, have focused on the demonstration of chemical self-reproduction under specific conditions. While several recent experimental studies have raised the possibility of carrying out some aspects of experimental evolution using autocatalytic reaction networks, there remain many open challenges. In this review, we start by evaluating theoretical studies of ACSs specifically with a view to establish the conditions required for such chemical systems to exhibit self-reproduction and Darwinian evolution. Then, we follow with an extensive overview of experimental ACS systems and use the theoretically established conditions to critically evaluate these empirical systems for their potential to exhibit Darwinian evolution. We identify various technical and conceptual challenges limiting experimental progress and, finally, conclude with some remarks about open questions.
... [1] To date, a diverse range of DNAzymes have been isolated to catalyze various chemical and biological transformations from DNA/RNA cleavage, ligation and phosphorylation to porphyrin metalation and peroxidation. [2][3][4][5][6][7][8][9] Since most natural DNA molecules are double-stranded, no catalytic activities are expected from them, and DNAzymes have so far only been isolated from in vitro selections. ...
DNAzymes are in vitro selected DNA oligonucleotides with catalytic activities. RNA cleavage is one of the most extensively studied DNAzyme reactions. To expand the chemical functionality of DNA, various chemical modifications have been made during and after selection. In this review, we summarize examples of RNA-cleaving DNAzymes and focus on those modifications introduced during in vitro selection. By incorporating various modified nucleotides via polymerase chain reaction (PCR) or primer extension, a few DNAzymes were obtained that can be specifically activated by metal ions such as Zn2+ and Hg2+. In addition, some modifications were introduced to mimic RNase A that can cleave RNA substrates in the absence of divalent metal ions. In addition, single modifications at the fixed regions of DNA libraries, especially at the cleavage junctions, have been tested, and examples of DNAzymes with phosphorothioate and histidine-glycine modified tertiary amine were successfully obtained specific for Cu2+, Cd2+, Zn2+, and Ni2+. Labeling fluorophore/quencher pair right next to the cleavage junction was also used to obtain signaling DNAzymes for detecting various metal ions and cells. Furthermore, we reviewed work on the cleavage of 2'-5' linked RNA and L-RNA substrates. Finally, applications of these modified DNAzymes as biosensors, RNases, and biochemical probes are briefly described with a few future research opportunities outlined at the end.
... All DNAzymes were isolated using a combinatorial method called in vitro selection. [15,18] Metal ions are indispensable for DNAzyme catalysis. [6,[19][20][21][22][23] DNA is highly negatively charged and metal ions can stabilize various secondary structures of DNA. ...
Since 1994, deoxyribozymes or DNAzymes have been in vitro selected to catalyze various types of reactions. Metal ions play a critical role in DNAzyme catalysis, and Zn²⁺ is a very important one among them. Zn²⁺ has good biocompatibility and can be used for intracellular applications. Chemically, Zn²⁺ is a Lewis acid and it can bind to both the phosphate backbone and the nucleobases of DNA. Zn²⁺ undergoes hydrolysis even at neutral pH, and the partially hydrolyzed polynuclear complexes can affect the interactions with DNA. These features have made Zn²⁺ a unique cofactor for DNAzyme reactions. This review summarizes Zn²⁺‐dependent DNAzymes with an emphasis on RNA‐/DNA‐cleaving reactions. A key feature is the sharp Zn²⁺ concentration and pH‐dependent activity for many of the DNAzymes. The applications of these DNAzymes as biosensors for Zn²⁺, as therapeutic agents to cleave intracellular RNA, and as chemical biology tools to manipulate DNA are discussed. Future studies can focus on the selection of new DNAzymes with improved performance and detailed biochemical characterizations to understand the role of Zn²⁺, which can facilitate practical applications of Zn²⁺‐dependent DNAzymes.
... A second approach is the directed experimental evolution of individual proteins or RNA molecules. Here, populations of molecules undergo repeated cycles of mutation and selection for a desired phenotype, such as the ability to cleave an antibiotic [39][40][41][42][43][44][45][46][47][48]. Directed evolution can proceed in vitro, in vivo, or in both. ...
Two main lines of research link information theory to evolutionary biology. The first focuses on organismal phenotypes , and on the information that organisms acquire about their environment. The second connects information-theoretic concepts to genotypic change. The genotypic and phenotypic level can be linked by experimental high-throughput genotyping and computational models of genotype-phenotype relationships. I here use a simple information-theoretic framework to compute a phenotype’s information content (its phenotypic complexity), and the information gain or change that comes with a new phenotype. I apply this framework to experimental data on DNA-binding phenotypes of multiple transcription factors. Low phenotypic complexity is associated with a biological system’s ability to discover novel phenotypes in evolution. I show that DNA duplications lower phenotypic complexity, which illustrates how information theory can help explain why gene duplications accelerate evolutionary adaptation. I also demonstrate that with the right experimental design, sequencing data can be used to infer the information gain associated with novel evolutionary adaptations, for example in laboratory evolution experiments. Information theory can help quantify the evolutionary progress embodied in the discovery of novel adaptive phenotypes.
... 221 Since then, numerous examples of RNA-cleaving DNA enzymes have been reported, all of them derived from synthetic DNA libraries. 222,223 Up to now, no natural catalytically active DNA has been found. RNA-cleaving DNA enzymes are practically useful tools for fundamental biochemical research, 224 and deoxyribozymes have been explored as mRNA-targeting agents for the downregulation of disease-relevant genes (before the advent of other potent gene regulation approaches such as RNAi and CRISPR/Cas strategies). ...
This review aims at juxtaposing common versus distinct structural and functional strategies that are applied by aptamers, riboswitches, and ribozymes/DNAzymes. Focusing on recently discovered systems, we begin our analysis with small-molecule binding aptamers, with emphasis on in vitro-selected fluorogenic RNA aptamers and their different modes of ligand binding and fluorescence activation. Fundamental insights are much needed to advance RNA imaging probes for detection of exo- and endogenous RNA and for RNA process tracking. Secondly, we discuss the latest gene expression-regulating mRNA riboswitches that respond to the alarmone ppGpp, to PRPP, to NAD+, to adenosine and cytidine diphosphates, and to precursors of thiamine biosynthesis (HMP-PP), and we outline new subclasses of SAM and tetrahydrofolate-binding RNA regulators. Many riboswitches bind protein enzyme cofactors that, in principle, can catalyse a chemical reaction. For RNA, however, only one system (glmS ribozyme) has been identified in Nature thus far that utilizes a small molecule - glucosamine-6-phosphate - to participate directly in reaction catalysis (phosphodiester cleavage). We wonder why that is the case and what is to be done to reveal such likely existing cellular activities that could be more diverse than currently imagined. Thirdly, this brings us to the four latest small nucleolytic ribozymes termed twister, twister-sister, pistol, and hatchet as well as to in vitro selected DNA and RNA enzymes that promote new chemistry, mainly by exploiting their ability for RNA labelling and nucleoside modification recognition. Enormous progress in understanding the strategies of nucleic acids catalysts has been made by providing thorough structural fundaments (e.g. first structure of a DNAzyme, structures of ribozyme transition state mimics) in combination with functional assays and atomic mutagenesis.
... Although some aptamers exist naturally as the binding domain of riboswitches (Doudna and Cech, 2002), most are generated by in vitro selection or SELEX (systematic evolution of ligands by exponential enrichment) (Wilson and Szostak, 1999). Similar to natural selection, in vitro selection is a Darwinian evolution process in which a large population of nucleic acid molecules (typically >10 14 unique sequences) is challenged to bind a target (Joyce, 2004). Molecules that bind to the target are recovered and amplified to generate a new population of molecules that has become enriched in members with the desired activity. ...
DNA polymerases play a central role in biology by transferring genetic information from one generation to the next during cell division. Harnessing the power of these enzymes in the laboratory has fueled an increase in biomedical applications that involve the synthesis, amplification, and sequencing of DNA. However, the high substrate specificity exhibited by most naturally occurring DNA polymerases often precludes their use in practical applications that require modified substrates. Moving beyond natural genetic polymers requires sophisticated enzyme-engineering technologies that can be used to direct the evolution of engineered polymerases that function with tailor-made activities. Such efforts are expected to uniquely drive emerging applications in synthetic biology by enabling the synthesis, replication, and evolution of synthetic genetic polymers with new physicochemical properties.
We present a method to create DNA-modified Prussian blue nanozymes, enhancing the stability and multi-analyte detection.
We used in vitro selection to identify DNAzymes that acylate the exocyclic nucleobase amines of cytidine, guanosine, and adenosine in DNA oligonucleotides. The acyl donor was the 2,3,5,6‐tetrafluorophenyl ester (TFPE) of a 5′‐carboxyl oligonucleotide. Yields are as high as >95% in 6 h. Several of the N‐acylation DNAzymes are catalytically active with RNA rather than DNA oligonucleotide substrates, and eight of nine DNAzymes for modifying C are site‐specific (>95%) for one particular substrate nucleotide. These findings expand the catalytic ability of DNA to include site‐specific N‐acylation of oligonucleotide nucleobases. Future efforts will investigate the DNA and RNA substrate sequence generality of DNAzymes for oligonucleotide nucleobase N‐acylation, toward a universal approach for site‐specific oligonucleotide modification.
We used in vitro selection to identify DNAzymes that acylate the exocyclic nucleobase amines of cytidine, guanosine, and adenosine in DNA oligonucleotides. The acyl donor was the 2,3,5,6‐tetrafluorophenyl ester (TFPE) of a 5′‐carboxyl oligonucleotide. Yields are as high as >95 % in 6 h. Several of the N‐acylation DNAzymes are catalytically active with RNA rather than DNA oligonucleotide substrates, and eight of nine DNAzymes for modifying C are site‐specific (>95 %) for one particular substrate nucleotide. These findings expand the catalytic ability of DNA to include site‐specific N‐acylation of oligonucleotide nucleobases. Future efforts will investigate the DNA and RNA substrate sequence generality of DNAzymes for oligonucleotide nucleobase N‐acylation, toward a universal approach for site‐specific oligonucleotide modification.
Amines can be alkylated using various reactions, such as reductive amination of aldehydes. In this study, we sought DNAzymes as catalytic DNA sequences that promote reductive amination with aliphatic amines, including DNA-anchored peptide substrates with lysine residues. By in vitro selection starting with either N40 or N20 random DNA pools, we identified many DNAzymes that catalyze reductive amination between the DNA oligonucleotide-anchored aliphatic amino group of DNA-C3-NH2 (C3 = short three-carbon tether) and a DNA-anchored benzaldehyde group in the presence of NaCNBH3 as reducing agent. At pH 5.2, 6.0, 7.5, or 9.0 in the presence of various divalent metal ion cofactors including Mg2+, Mn2+, Zn2+ and Ni2+, the DNAzymes have kobs up to 0.12 h-1 and up to 130-fold rate enhancement relative to the DNA-splinted but uncatalyzed background reaction. However, analogous selection experiments did not lead to any DNAzymes that function with DNA-HEG-NH2 [HEG = long hexa(ethylene glycol) tether], or with short- and long-tethered DNA-AAAKAA and DNA-HEG-AAAKAA lysine-containing hexapeptide substrates (A = alanine, K = lysine). Including a variety of other amino acids in place of the neighboring alanines also did not lead to DNAzymes. These findings establish a practical limit on the substrate scope of DNAzyme catalysis for N-alkylation of aliphatic amines by reductive amination. The lack of DNAzymes for reductive amination with any substrate more structurally complex than DNA-C3-NH2 is likely related to the challenge in binding and spatially organizing those other substrates. Because other reactions such as aliphatic amine N-acylation are feasible for DNAzymes with DNA-anchored peptides, our findings show that the ability to identify DNAzymes depends strongly on both the investigated reaction and the composition of the substrate.
DNA nanotechnology relies on the structural and functional information encoded in nucleic acids. Specifically, the sequence‐guided reconfiguration of nucleic acids by auxiliary triggers provides a means to develop DNA switches, machines and stimuli‐responsive materials. The present Review addresses recent advances in the construction and applications of dynamic reconfigurable DNA nanostructures, networks and materials. Dynamic transformations proceeding within engineered origami frames or between origami tiles, and the triggered dynamic reconfiguration of scaled supramolecular origami structures are addressed. The use of origami frameworks to assemble dynamic chiroplasmonic optical devices and to operate switchable chemical processes are discussed. Also, the dynamic operation of DNA networks is addressed, and the design of “smart” stimuli‐responsive all‐DNA materials and their applications are introduced. Future perspectives and applications of dynamic reconfigurable DNA nanostructures are presented.
DNA nanotechnology relies on the structural and functional information encoded in nucleic acids. Specifically, the sequence‐guided reconfiguration of nucleic acids by auxiliary triggers provided means to develop DNA switches, machines and stimuli‐responsive materials. The present review article addresses recent advances in the constructions and applications of dynamic reconfigurable DNA nanostructures, networks and materials. Dynamic transformations proceeding within engineered origami frames or between origami tiles, and the triggered dynamic reconfiguration of scaled supramolecular origami structures are addressed. The use of origami frameworks as playground to assemble dynamic chiroplasmonic optical devices and to operate switchable chemical processes are discussed. Also, the dynamic operation of DNA networks are addressed, and the design of “smart” stimuli‐responsive all‐DNA materials and their applications are introduced. Future perspectives and applications of dynamic reconfigurable DNA nanostructures are presented.
In the past few years, with the in-depth research of functional nucleic acids and isothermal amplification techniques, their applications in the field of biosensing have attracted great interest. Since functional nucleic acids have excellent flexibility and convenience in their structural design, they have significant advantages as recognition elements in biosensing. At the same time, isothermal amplification techniques have higher amplification efficiency, so the combination of functional nucleic acids and isothermal amplification techniques can greatly promote the widespread application of biosensors. For the purpose of further improving the performance of biosensors, this review introduces several widely used functional nucleic acids and isothermal amplification techniques, as well as their classification, basic principles, application characteristics, and summarizes their important applications in the field of biosensing. We hope to provide some references for the design and construction of new tactics to enhance the detection sensitivity and detection range of biosensing.
Photoresponsive nucleic acids attract growing interest as functional constituents in materials science. Integration of photoisomerizable units into DNA strands provides an ideal handle for the reversible reconfiguration of nucleic acid architectures by light irradiation, triggering changes in the chemical and structural properties of the nanostructures that can be exploited in the development of photoresponsive functional devices such as machines, origami structures and ion channels, as well as environmentally adaptable 'smart' materials including nanoparticle aggregates and hydrogels. Moreover, photoresponsive DNA components allow control over the composition of dynamic supramolecular ensembles that mimic native networks. Beyond this, the modification of nucleic acids with photosensitizer functionality enables these biopolymers to act as scaffolds for spatial organization of electron transfer reactions mimicking natural photosynthesis. This review provides a comprehensive overview of these exciting developments in the design of photoresponsive DNA materials, and showcases a range of applications in catalysis, sensing and drug delivery/release. The key challenges facing the development of the field in the coming years are addressed, and exciting emergent research directions are identified.
The last decade has witnessed tremendous growth in the field of synthetic genetics, an area of synthetic biology that applies concepts that are commonly associated with the field of genetics, such as heredity and evolution, to artificial genetic polymers with novel backbone structures (XNAs). In addition to the emergence of biologically stable affinity reagents (aptamers), progress in this area has led to the discovery of XNA enzymes (XNAzymes) that are capable of mediating transphosphorylation chemistry with multiple turnover activity. This review explores the evolution and rational design of XNAzymes as well as their potential as reagents in biomedical applications.
Ewolucja darwinowska nie jest synonimem zmiany, lecz wyjątkowym procesem biologicznym. Biochemiczny mechanizm ewolucji jest inny niż wynikało to z obserwacji Darwina dotyczących dziedzicznej zmienności i doboru naturalnego. Kluczem do ewolucji biologicznej jest ścisły związek między dziedziczonym genotypem a zależnym od genów fenotypem. Dzięki temu związkowi fenotyp może stanowić przedmiot selekcji. Jest teoretycznie możliwe, by pewne formy życia nie podlegały ewolucji. Pochodzenie życia i pochodzenie ewolucji to dwa odrębne problemy badawcze. Klasyczny problem teleologii w biologii da się rozwiązać dzięki starannemu zbadaniu mechanizmu odpowiadającego za związek między genotypem a fenotypem, czyli mechanizmu syntezy białek lub systemu translacji. Ten mechanizm przekształcania chemii kwasów nukleinowych w chemię białek może stanowić fundamentalne źródło teleonomii i wewnętrznej teleologii w organizmach żywych.
Catalytic DNA molecules (DNAzymes) have joined the ranks of biopolymers capable of catalyzing chemical transformations. Unlike their RNA and protein counterparts, naturally occurring DNAzymes have never been identified and are man‐made molecules obtained by a Darwinian in vitro evolution method coined SELEX (Systematic Evolution of Ligands by Exponential Enrichment). This chapter introduces the reader to the world of DNAzymes and describes their catalytic repertoire and highlights their usefulness in numerous practical applications such as gene therapy or metal biosensing. This chapter also presents mechanistic and structural insights into the modus operandi of these biocatalysts. Despite their intrinsic properties, DNAzymes present some shortcomings including poor cellular uptake, low residence times and poor pharmacokinetic properties, and limited access to functional groups to mediate catalysis. Hence, a last section of this chapter is dedicated to discussing the introduction of chemical modifications into the scaffold of DNAzymes to alleviate these limitations.
DNAzymes are synthetic DNA molecules that catalyze chemical reactions with the help of metal ion cofactors. Compared to protein enzymes and ribozymes, DNAzymes have advantages such as high stability, low cost, and good programmability. Over the last decade, RNA‐cleaving DNAzymes have been widely utilized as biosensors for a diverse range of analytes, including metal ions, small molecules, and proteins. In particular, the sensitivity and selectivity of their catalytic activity toward metal ions are ideal for metal ion detection. In this chapter, we introduce several representative DNAzymes according to their binding targets. Various DNAzyme‐based biosensors displaying fluorescent, colorimetric, or electrochemical signals are also discussed. Furthermore, signal amplification methods developed to improve the sensitivity of DNAzyme‐based sensors are described.
Directed evolution is a vital approach in generating novel therapeutics and biotechnological tools. The three key steps in any directed evolution campaign are diversification of genetic material, translation into functional biomolecules, and selection of fitter variants. The emerging field of continuous directed evolution aims to streamline these steps with engineered systems that require little or no manual staging of the evolutionary process. A shared principle behind these designs is the achievement of genetic systems with exceptionally high mutation rates targeted only to the DNA that encodes the evolving biomolecule of interest while maintaining a normal mutation rate in other DNA. Such targeted rapid mutagenesis technologies support continuous evolution of biomolecules. The systems that we review in this chapter are highly useful in reducing the time and effort required for evolving a wide range of biomolecules. In addition, the evolution systems considered facilitate deeper explorations of sequence space at extensive scale, a consequence of the speed and parallelization of directed evolution enabled by continuous evolution. Continuous evolution therefore increases the likelihood of success in directed evolution campaigns and opens up new areas of investigation, including reaching ambitious biomolecular functions that require long mutational pathways and greater understanding of biomolecular sequence–function relationships.
The assembly of DNA with metal-complex cofactors can form promising biocatalysts for asymmetric reactions, although catalytic performance is typically limited by low enantioselectivities and stereo-control remains a challenge. Here, we engineer G-quadruplex-based DNA biocatalysts for an asymmetric cyclopropanation reaction, achieving enantiomeric excess (eetrans) values of up to +91% with controllable stereoinversion, where the enantioselectivity switches to -72% eetrans through modification of the Fe-porphyrin cofactor. Complementary circular dichroism, nuclear magnetic resonance, and fluorescence titration experiments show that the porphyrin ligand of the cofactor participates in the regulation of the catalytic enantioselectivity via a synergetic effect with DNA residues at the active site. These findings underline the important role of cofactor modification in DNA catalysis and thus pave the way for the rational engineering of DNA-based biocatalysts.
Metalloporphyrins play important roles in biology, such as magnesium porphyrin for photosynthesis and iron porphyrin for carrying and transferring oxygen. They are also powerful molecules for the development of biosensors, phototherapy, photocatalysis, photodegradation, light harvesting, and water splitting. However, the porphyrin metalation reaction is difficult to achieve at room temperature due to a high kinetic barrier. Inspired by the pioneering work in catalytic antibodies, ribozymes, DNAzymes and nanozymes have been developed as enzyme mimics to accelerate this reaction. This review summarizes the progress in DNAzymes and nanozymes due to their excellent stability and low cost. We first introduce the structure and property of common porphyrins and metalloporphyrins. DNAzymes for porphyrin metalation are then reviewed, including early work, recent work using Pb2+ as a cofactor, and non-G-quadruplex DNAzymes. The catalytic mechanisms of DNAzymes are also discussed, especially the role of metal ions. Subsequently, nanozymes for porphyrin metalation based on graphene and a few other nanomaterials are reviewed. In this part, the interactions between the nanozymes and porphyrins are elucidated to describe the catalytic effect. In addition, beta-cyclodextrin and some surfactants that can form micelles in water were also found to have catalytic activity. Finally, we review the applications of porphyrin metalation reactions for the detection of various metal ions, improving photocatalytic activity, and removing heavy metal ions in water.
Highly sensitive and selective detection of lanthanide ions is a major analytical challenge. In recent years, the use of DNA for this purpose has been pursued. For such highly charged cations, it is difficult to select their aptamers due to strong nonspecific binding. On the other hand, the use of catalytic DNA or DNAzymes has an advantage to overcome this problem, especially DNAzymes with RNA-cleaving activity. In this chapter, a few such DNAzymes are introduced and methods for in vitro selection of lanthanide-dependent RNA-cleaving DNAzymes are described in detail, including the selection protocols, the DNA sequences used, the characterization of selected DNAzymes and their conversion into biosensors. All of the experiments use only fluorophore-labeled DNA, and radioisotope labeling is completely avoided. The resulting DNAzymes can distinguish lanthanides from non-lanthanide metals, tell the difference between light and heavy lanthanides, and can be used together to discriminate individual lanthanides.
Sie enthalten zwei oder mehr verschiedene Typen an Monomereinheiten.
DNAzymes were previously identified by in vitro selection for a variety of chemical reactions, including several biologically relevant peptide modifications. However, finding DNAzymes for peptide lysine acylation is a substantial challenge. By using suitably reactive aryl ester acyl donors as the electrophiles, here we used in vitro selection to identify DNAzymes that acylate amines, including lysine side chains of DNA-anchored peptides. Some of the DNAzymes can transfer a small glutaryl group to an amino group. These results expand the scope of DNAzyme catalysis and suggest the future broader applicability of DNAzymes for sequence-selective lysine acylation of peptide and protein substrates.
Phosphorothioate (PS) modification replaces one of the non-bridging oxygen atoms by sulfur in the phosphate backbone of nucleic acids. While PS DNAs have been traditionally used as nuclease-resistant antisense agents and PS RNA as probe of metal binding in ribozymes, multiple new applications have emerged in recent years. In this review, we start by briefly introducing the structure and synthesis of PS nucleic acids followed by their fundamental chemical and biochemical properties. Further, their recently emerged surface science applications are discussed, such as attachment of DNA to various surfaces and nanomaterials containing thiophilic metals such as gold, silver and cadmium, and templating the growth of these materials. Their role in conferring structural effects in the presence of certain metal ions and in fishing out novel aptamers are also discussed. Covalent chemistry can be performed on the sulfur atom for further grafting functional groups to the backbone of DNA. For PS RNA, we discuss their role as probes for metal binding in ribozymes and DNAzymes, which leads to applications in detection of thiophilic metal ions. Since each PS modification site produces a chiral phosphorus center, the synthesis and purification of diastereomers and their applications are emphasized throughout this review. In the end, a few future research directions are discussed.
Within the broad research efforts to engineer chemical pathways to yield high-throughput evolutionary synthesis of genes and their screening for dictated functionalities, we introduce the evolution of nucleic-acid-based constitutional dynamic networks (CDNs) that follows reproduction/variation/selection principles. These fundamental principles are demonstrated by assembling a library of nucleic-acid strands and hairpins as functional modules for evolving networks. Primary T1-initiated selection of components from the library assembles a parent CDN X, where the evolved constituents exhibit catalytic properties to cleave the hairpins in the library. Cleavage of the hairpins yields fragments, which reproduces T1 to replicate CDN X, whereas the other fragments T2 and T3 select other components to evolve two other CDNs, Y and Z (variation). By applying appropriate counter triggers, we demonstrate the guided selection of networks from the evolved CDNs. By integrating additional hairpin substrates into the system, CDN-dictated emergent catalytic transformations are accomplished. The study provides pathways to construct evolutionary dynamic networks revealing enhanced gated and cascaded functions.
Fundamentally, riboswitches are known to be highly structured and conserved metabolite binding domains, known to be aptamer, which are locus within mRNAs. When we talk about RNA world, riboswitches are ranked among the trending topics. Since its discovery from 2002, there is a large amount of feature disclosed till now and thanks to computational analysis this finding are increasing every year with a fast rate. This work club up the information regarding new findings within the field of riboswitches, which include data about its new classes discovery and its application in the field of gene regulation mechanism, medical science, inter-cellular signalling, biosensors and many more.
A method to assemble loaded stimuli-responsive DNA-polyacrylamide hydrogel-stabilized microcapsules is presented. The method involves the coating of substrate-loaded CaCO3 microparticles, functionalized with nucleic acid promoter units, and the crosslinking of DNA-modified polyacrylamide chains, on the microcapsules, using the hybridization chain reaction (HCR), to yield the DNA-crosslinked hydrogel coating. Dissolution of the CaCO3 particles generated the substrate-loaded hydrogel protected microcapsules. The microcapsule-hydrogel shells include engineered stimuli-responsive oligonucleotides crosslinkers that control the stiffness of the hydrogel shells, allowing the triggered release of the loads. One approach includes the incorporation of cofactor-dependent DNAzyme units into the crosslinked hydrogel layer (cofactor = Mg²⁺-ions, Zn²⁺-ions or histidine) as stimuli-responsive units. Cleavage of the crosslinking DNAzyme substrates by the respective cofactors yields hydrogel coatings of reduced stiffness and higher porosity, that allows the release of the loads. A further approach has involved the application of the HCR process to assemble the bi-layer hydrogel microcapsules being unlocked by two cooperative triggers. Bi-layer microcapsules consisting of a K⁺-ions stabilized G-quadruplex/18-crown-6-ether responsive layer and a Mg²⁺-ion DNAzyme responsive layer is presented. Unlocking and locking of the G-quadruplex crosslinked layer by 18-crown-6-ether and K⁺-ions, respectively, and in the presence of Mg²⁺-ions, allows switchable controlled release of the load. In addition, the intercommunication of two kinds of stimuli-responsive bi-layer hydrogel microcapsules carrying two different loads (Tetramethylrhodamine-dextran, TMR-D, and CdSe/ZnS quantum dots) is demonstrated. The intercommunication process involves the stimuli-triggered generation of “information transfer” strands from one microcapsule to another that activate the release of the loads.
Like most proteins, complex RNA molecules often are modular objects made up of distinct structural and functional domains.
The component domains of a protein can associate in alternative combinations to form molecules with different functions. These
observations raise the possibility that complex RNAs also can be assembled from preexisting structural and functional domains.
To test this hypothesis, an in vitro evolution procedure was used to isolate a previously undescribed class of complex ligase ribozymes, starting from a pool
of 1016 different RNA molecules that contained a constant region derived from a large structural domain that occurs within self-splicing
group I ribozymes. Attached to this constant region were three hypervariable regions, totaling 85 nucleotides, that gave rise
to the catalytic motif within the evolved catalysts. The ligase ribozymes catalyze formation of a 3′,5′-phosphodiester linkage
between adjacent template-bound oligonucleotides, one bearing a 3′ hydroxyl and the other a 5′ triphosphate. Ligation occurs
in the context of a Watson–Crick duplex, with a catalytic rate of 0.26 min−1 under optimal conditions. The constant region is essential for catalytic activity and appears to retain the tertiary structure
of the group I ribozyme. This work demonstrates that complex RNA molecules, like their protein counterparts, can share common
structural domains while exhibiting distinct catalytic functions.
The RNA of viroids and virusoids in plants, and the RNA transcripts of some tandemly repeated DNA sequences in the newt, can undergo self-catalysed cleavage to generate RNA with 5'-OH and 2',3'-cyclic-phosphate termini. These catalytic RNAs, or ribozymes, form a stem-loop secondary structure called a 'hammerhead' in which the catalytic (ribozyme) and substrate sequences are brought close together. Catalytically active mimics of hammerhead ribozymes can be readily made using oligoribonucleotides. Consequently, hammerhead analogues in which certain ribonucleotides are replaced by different ones have been constructed both to identify consensus residues required for cleavage activity and to determine the details of the cleavage mechanism. But these ribonucleotide-replacements tend to alter the conformation of the hammerhead by changing hydrogen-bonding and stacking potential at the position of substitution. We have now constructed structurally less-disrupted hammerhead analogues in which deoxyribonucleotides, which lack 2'-OH groups, are substituted for ribonucleotides. These mixed RNA-DNA polymers were synthesized using a strategy for the chemical synthesis of RNA that is compatible with DNA synthesis. Analysis of the cleavage products of several of these hammerhead analogues confirms the involvement in the reaction of the 2'-OH adjacent to the cleavage site in the substrate, and demonstrates that some 2'-OH groups in the catalytic region strongly affect activity. The results also indicate that the three-dimensional structure producing nucleic acid-type catalysis is not restricted to RNA.
A target nucleic acid sequence can be replicated (amplified) exponentially in vitro under isothermal conditions by using three enzymatic activities essential to retroviral replication: reverse transcriptase, RNase H, and a DNA-dependent RNA polymerase. By mimicking the retroviral strategy of RNA replication by means of cDNA intermediates, this reaction accumulates cDNA and RNA copies of the original target. Product accumulation is exponential with respect to time, indicating that newly synthesized cDNAs and RNAs function as templates for a continuous series of transcription and reverse transcription reactions. Ten million-fold amplification occurs after a 1- to 2-hr incubation, with an initial rate of amplification of 10-fold every 2.5 min. This self-sustained sequence replication system is useful for the detection and nucleotide sequence analysis of rare RNAs and DNAs. The analogy to aspects of retroviral replication is discussed.
The structure and replication of the single-stranded circular RNA genome of hepatitis delta virus (HDV) are unique relative to those of known animal viruses, and yet there are real similarities between HDV and certain infectious RNAs of plants. Therefore, since some of the latter RNAs have been shown to undergo in vitro site-specific cleavage and even ligation, we tested the hypothesis that similar events might also occur for HDV RNA. In partial confirmation of this hypothesis, we found that in vitro the RNA complementary to the HDV genome, the antigenomic RNA, could undergo a self-cleavage that was not only more than 90% efficient but also occurred only at a single location. This cleavage was found to produce junction fragments consistent with a 5'-hydroxyl and a cyclic 2',3'-monophosphate. Since the observed cleavage was both site-specific and occurred only once per genome length, we propose that the site may be relevant to the normal intracellular replication of the HDV genome. Because the site is located almost adjacent to the 3' end of the delta antigen-coding region, the only known functional open reading frame of HDV, we suggest that the cleavage may have a role not only in genome replication but also in RNA processing, helping to produce a functional mRNA for the translation of delta antigen.
The RNA moieties of ribonuclease P purified from both E. coli (M1 RNA) and B. subtilis (P-RNA) can cleave tRNA precursor molecules in buffers containing either 60 mM Mg2+ or 10 mM Mg2+ plus 1 mM spermidine. The RNA acts as a true catalyst under these conditions whereas the protein moieties of the enzymes alone show no catalytic activity. However, in buffers containing 5-10 mM Mg2+ (in the absence of spermidine) both kinds of subunits are required for enzymatic activity, as shown previously. In the presence of low concentrations of Mg2+, in vitro, the RNA and protein subunits from one species can complement subunits from the other species in reconstitution experiments. When the precursor to E. coli 4.5S RNA is used as a substrate, only the enzyme complexes formed with M1 RNA from E. coli and the protein moieties from either bacterial species are active.
A study of the activity of deoxyribonucleotide-substituted analogs of the hammerhead domain of RNA catalysis has led to the
design of a 14mer oligomer composed entirely of deoxyribonucleotides that promotes the cleavage of an RNA substrate. Characterization
of this reaction with sequence variants and mixed DNA/RNA oligomers shows that, although the all-deoxyribonucleotide oligomer
is less efficient in catalysis, the DNA/substrate complex shares many of the properties of the all-RNA hammerhead domain such
as multiple turnover kinetics and dependence on Mg2+ concentration. On the other hand, the values of kinetic parameters distinguish the DNA oligomer from the all-RNA oligomer.
In addition, an analog of the oligomer having a single ribonucleotide in a strongly conserved position of the hammerhead domain
is associated with more efficient catalysis than the all-RNA oligomer.
Seven families of RNA ligases, previously isolated from random RNA sequences, fall into three classes on the basis of secondary structure and regiospecificity of ligation. Two of the three classes of ribozymes have been engineered to act as true enzymes, catalyzing the multiple-turnover transformation of substrates into products. The most complex of these ribozymes has a minimal catalytic domain of 93 nucleotides. An optimized version of this ribozyme has a kcat exceeding one per second, a value far greater than that of most natural RNA catalysts and approaching that of comparable protein enzymes. The fact that such a large and complex ligase emerged from a very limited sampling of sequence space implies the existence of a large number of distinct RNA structures of equivalent complexity and activity.
An iterative in vitro selection procedure was used to isolate a new class of catalytic RNAs (ribozymes) from a large pool
of random-sequence RNA molecules. These ribozymes ligate two RNA molecules that are aligned on a template by catalyzing the
attack of a 3'-hydroxyl on an adjacent 5'-triphosphate--a reaction similar to that employed by the familiar protein enzymes
that synthesize RNA. The corresponding uncatalyzed reaction also yields a 3',5'-phosphodiester bond. In vitro evolution of
the population of new ribozymes led to improvement of the average ligation activity and the emergence of ribozymes with reaction
rates 7 million times faster than the uncatalyzed reaction rate.
We have synthesized 13 hammerhead ribozyme variants, each containing an abasic residue at a specific position of the catalytic core. The activity of each of the variants is significantly reduced. In four cases, however, activity can be rescued by exogenous addition of the missing base. For one variant, the rescue is 300-fold; for another, the rescue is to the wild-type level. This latter abasic variant (G10.1X) has been characterized in detail. Activation is specific for guanine, the base initially removed. In addition, the specificity for guanine versus adenine is substantially altered by replacing C with U in the opposite strand of the ribozyme. These results show that a binding site for a small, noncharged ligand can be created in a preexisting ribozyme structure. This has implications for structure-function analysis of RNA, and leads to speculations about evolution in an "RNA world" and about the potential therapeutic use of ribozymes.
With the eventual goal of developing a treatment for chronic myelogenous leukemia (CML), attempts have been made to design
hammerhead ribozymes that can specifically cleave BCR-ABL fusion mRNA. In the case of L6 BCR-ABL fusion mRNA (b2a2 type; BCR exon 2 is fused to ABL exon 2), which has no effective cleavage sites for conventional hammerhead ribozymes near the BCR-ABL junction, it has proved very difficult to cleave the chimeric mRNA specifically. Several hammerhead ribozymes with relatively
long junction-recognition sequences have poor substrate-specificity. Therefore, we explored the possibility of using newly
selected DNA enzymes that can cleave RNA molecules with high activity to cleave L6 BCR-ABL fusion (b2a2) mRNA. In contrast to the results with the conventional ribozymes, the newly designed DNA enzymes, having higher
flexibility for selection of cleavage sites, were able to cleave this chimeric RNA molecule specifically at sites close to
the junction. Cleavage occurred only within the abnormal BCR-ABL mRNA, without any cleavage of the normal ABL or BCR mRNA. Thus, these chemically synthesized DNA enzymes seem to be potentially useful for application in vivo, especially for the treatment of CML, if we can develop exogenous delivery strategies.
Background:
Efficient operation of cellular processes relies on the strict control that each cell exerts over its metabolic pathways. Some protein enzymes are subject to allosteric regulation, in which binding sites located apart from the enzyme's active site can specifically recognize effector molecules and alter the catalytic rate of the enzyme via conformational changes. Although RNA also performs chemical reactions, no ribozymes are known to operate as true allosteric enzymes in biological systems. It has recently been established that small-molecule receptors can readily be made of RNA, as demonstrated by the in vitro selection of various RNA aptamers that can specifically bind corresponding ligand molecules. We set out to examine whether the catalytic activity of an existing ribozyme could be brought under the control of an effector molecule by designing conjoined aptamer-ribozyme complexes.
Results:
By joining an ATP-binding RNA to a self-cleaving ribozyme, we have created the first example of an allosteric ribozyme that has a catalytic rate that can be controlled by ATP. A 180-fold reduction in rate is observed upon addition of either adenosine or ATP, but no inhibition is detected in the presence of dATP or other nucleoside triphosphates. Mutations in the aptamer domain that are expected to eliminate ATP binding or that increase the distance between aptamer and ribozyme domains result in a loss of ATP-specific allosteric control. Using a similar design approach, allosteric hammerhead ribozymes that are activated in the presence of ATP were created and another ribozyme that can be controlled by theophylline was created.
Conclusions:
The catalytic features of these conjoined aptamer-ribozyme constructs demonstrate that catalytic RNAs can also be subject to allosteric regulation-a key feature of certain protein enzymes. Moreover, by using simple rational design strategies, it is now possible to engineer new catalytic polynucleotides which have rates that can be tightly and specifically controlled by small effector molecules.
The hypothesis that certain RNA molecules may be able to catalyse RNA replication is central to current theories of the early evolution of life. In support of this idea, we describe here an RNA that synthesizes RNA using the same reaction as that employed by protein enzymes that catalyse RNA polymerization. In the presence of the appropriate template RNA and nucleoside triphosphates, the ribozyme extends an RNA primer by successive addition of up to six mononucleotides. The added nucleotides are joined to the growing RNA chain by 3',5'-phosphodiester linkages. The ribozyme shows marked template fidelity: extension by nucleotides complementary to the template is up to 1,000 times more efficient than is extension by mismatched nucleotides.
A group of highly efficient Zn(II)-dependent RNA-cleaving deoxyribozymes has been obtained through in vitro selection. They share a common motif with the ‘8–17’ deoxyribozyme isolated under different conditions, including different
design of the random pool and metal ion cofactor. We found that this commonly selected motif can efficiently cleave both RNA
and DNA/RNA chimeric substrates. It can cleave any substrate containing rNG (where rN is any ribonucleotide base and G can
be either ribo- or deoxyribo-G). The pH profile and reaction products of this deoxyribozyme are similar to those reported
for hammerhead ribozyme. This deoxyribozyme has higher activity in the presence of transition metal ions compared to alkaline
earth metal ions. At saturating concentrations of Zn2+, the cleavage rate is 1.35 min–1 at pH 6.0; based on pH profile this rate is estimated to be at least ~30 times faster at pH 7.5, where most assays of Mg2+-dependent DNA and RNA enzymes are carried out. This work represents a comprehensive characterization of a nucleic acid-based
endonuclease that prefers transition metal ions to alkaline earth metal ions. The results demonstrate that nucleic acid enzymes
are capable of binding transition metal ions such as Zn2+ with high affinity, and the resulting enzymes are more efficient at RNA cleavage than most Mg2+-dependent nucleic acid enzymes under similar conditions.
Small satellite RNAs1–5 of plant viruses depend on the presence of a supporting RNA virus for their propagation in vivo. Replication of the 359–nucleotide-long6 satellite RNA of tobacco ringspot virus (STobRV RNA) is detected only in tissue infected with tobacco ringspot virus (TobRV). STobRV RNA becomes encapsidated in TobRV coat protein and acts as a parasite of TobRV, reducing its accumulation and the severity of symptoms that it induces. Here we report the transcription in vitro of a circularly permuted, complementary DNA clone of STobRV RNA oriented so as to produce RNA that is complementary to the encapsidated, (+) polarity STobRV RNA. Like STobRV (+)RNA7, this dimeric, circularly permuted STobRV (−)RNA cleaves autolytically. Cleavage is at two identical sites generating monomeric-length RNA and two terminal fragments. The new termini are 5′-hydroxyl and 2′:3′-cyclic phosphodiester groups. The RNAs ligate spontaneously to give linear and, from the monomers, circular molecules. Replication of STobRV RNA may require these autoly-sis and ligation reactions, which, at least in vitro, occur without enzyme catalysis.
In vitro selection experiments yield unexpected results. An RNA-cleaving DNA enzyme that was selected in the presence of Mg2+ ions is much more efficient with Ca2+ as the cofactor. The result is even more surprising because of the lower hydrolytic efficiency of Ca2+ itself.
A model of an objective function based on polynucleotide folding is used to investigate the dynamics of evolutionary adaptation in finite populations. Binary sequences are optimized with respect to their kinetic properties through a stochastic process involving mutation and selection. The objective function consists in a mapping from the set of all binary strings with given length into a set of two-dimensional structures. These structures then encode the kinetic properties, expressed in terms of parameters of reaction probability distributions. The objective function obtained thereby represents a realistic example of a highly ``rugged landscape.'' Ensembles of molecular strings adapting to this landscape are studied by tracing their escape path from local optima and by applying multivariate analysis. Effects of small population numbers in the tail of the sequence distribution are discussed quantitatively. Close upper bounds to the number of distinct values produced by our objective function are given. The distribution of values is explored by means of simulated annealing and reveals a random scatter in the locations of optima in the space of all sequences. The genetic optimization protocol is applied to the ``traveling salesman'' problem.
A detailed understanding of the susceptibility of RNA phosphodiesters to specific base-catalyzed cleavage is necessary to approximate the stability of RNA under various conditions. In addition, quantifying the rate enhancements that can be produced exclusively by this common cleavage mechanism is needed to fully interpret the mechanisms employed by ribonucleases and by RNA-cleaving ribozymes. Chimeric DNA/RNA oligonucleotides were used to examine the rates of hydroxide-dependent degradation of RNA phosphodiesters under reaction conditions that simulate those of biological systems. Under neutral or alkaline pH conditions, the dominant pathway for RNA degradation is an internal phosphoester transfer reaction that is promoted by specific base catalysis. As expected, increasing the concentration of hydroxide ion, increasing the concentration of divalent magnesium, or raising the temperature accelerates strand scission. In most instances, the identities of the nucleotide bases that flank the target RNA linkage have a negligible effect on the pKa of the nucleophilic 2‘-hydroxyl group, and only have a minor effect on the maximum rate constant for the transesterification reaction. Under representative physiological conditions, specific base catalysis of RNA cleavage generates a maximum rate enhancement of 100 000-fold over the background rate of RNA transesterification. The kinetic parameters reported herein provide theoretical limits for the stability of RNA polymers and for the proficiency of RNA-cleaving enzymes and enzyme mimics that exclusively employ a mechanism of general base catalysis.
New DNA: By enzymatic polymerization of base-modified nucleoside triphosphates, a functionalized DNA (fDNA; see picture) was generated in which every base bears an additional amino acid like residue, thus mimicking the functional group repertoire of peptides on a nucleic acid backbone. These modified oligonucleotides can in turn serve as templates for polymerase chain reaction amplification, thus utilizing fDNA as a novel class of biopolymers for in vitro selection techniques.
The discovery of a simple structural motif allows for the enzymatic synthesis by polymerase chain reactions (PCR) of modified DNA (see reaction scheme) bearing side chains similar or even identical to those of several amino acids. Libraries of DNA functionalized with both cationic and anionic groups may now be readily prepared. R=I, HgCl; X=functional group.
We have constructed catalytic molecular beacons from a hammerhead-type deoxyribozyme by a modular design. The deoxyribozyme was engineered to contain a molecular beacon stem–loop module that, when closed, inhibits the deoxyribozyme module and is complementary to a target oligonucleotide. Binding of target oligonucleotides opens the beacon stem–loop and allosterically activates the deoxyribozyme module, which amplifies the recognition event through cleavage of a doubly labeled fluorescent substrate. The customized modular design of catalytic molecular beacons allows for any two single-stranded oligonucleotide sequences to be distinguished in homogenous solution in a single step. Our constructs demonstrate that antisense conformational triggers based on molecular beacons can be used to initiate catalytic events. The selectivity of the system is sufficient for analytical applications and has potential for the construction of deoxyribozyme-based drug delivery tools specifically activated in cells containing somatic mutations.
A group of highly efficient Zn(II)-dependent RNA-cleaving deoxyribozymes has been obtained through in vitro selection. They share a common motif with the '8-17' deoxyribozyme isolated under different conditions, including different design of the random pool and metal ion cofactor. We found that this commonly selected motif can efficiently cleave both RNA and DNA/RNA chimeric substrates. It can cleave any substrate containing rNG (where rN is any ribo-nucleotide base and G can be either ribo- or deoxy-ribo-G). The pH profile and reaction products of this deoxyribozyme are similar to those reported for hammerhead ribozyme. This deoxyribozyme has higher activity in the presence of transition metal ions compared to alkaline earth metal ions. At saturating concentrations of Zn(2+), the cleavage rate is 1.35 min(-1)at pH 6.0; based on pH profile this rate is estimated to be at least approximately 30 times faster at pH 7.5, where most assays of Mg(2+)-dependent DNA and RNA enzymes are carried out. This work represents a comprehensive characterization of a nucleic acid-based endonuclease that prefers transition metal ions to alkaline earth metal ions. The results demonstrate that nucleic acid enzymes are capable of binding transition metal ions such as Zn(2+)with high affinity, and the resulting enzymes are more efficient at RNA cleavage than most Mg(2+)-dependent nucleic acid enzymes under similar conditions.
A modified polymerase chain reaction (PCR) was developed to introduce random point mutations into cloned genes. The modifications were made to decrease the fidelity of Taq polymerase during DNA synthesis without significantly decreasing the level of amplification achieved in the PCR. The resulting PCR products can be cloned to produce random mutant libraries or transcribed directly if a T7 promoter is incorporated within the appropriate PCR primer. We used this method to mutagenize the gene that encodes the Tetrahymena ribozyme with a mutation rate of 0.66% +/- 0.13% (95% C.I.) per position per PCR, as determined by sequence analysis. There are no strong preferneces with respect to the type of base substituion. The number of mutations per DNA sequence follows a Poisson distribution and the mutations are randomly distributed throughout the amplified sequence.
Several mixed DNA/RNA and 2'-O-methylribonucleotide/RNA analogues derived from the "hammerhead" domain of RNA catalysis have been prepared to study the minimum ribonucleotide requirement for catalytic activity. Oligodeoxyribonucleotides containing from seven to as few as four ribonucleotides are active in cleaving a substrate RNA. Predominantly deoxyribonucleotide-containing analogues have kcat values 20-300 and kcat/KM values approximately 100-2000 times lower than those of all-RNA ribozyme. In the case of predominantly 2'-O-methyl analogues, at least five ribonucleotides are needed to assure catalytic activity. In addition, both predominantly deoxyribonucleotide and 2'-O-methyl oligomers are at least 3 orders of magnitude more stable than an all-RNA ribozyme in incubations with RNase A and a yeast extract. These results suggest that the ribophosphate backbone is not a strict requirement for ribozyme-type catalysis. The identification of the four required ribonucleotides in the hammerhead catalytic domain provides valuable information for the rational design of chemical species having ribonuclease activities.
The use of deoxyribonucleotide substitution in RNA (mixed RNA/DNA polymers) permits an evaluation of the role of 2'-hydroxyl groups in ribozyme catalysis. Specific deoxyribonucleotide substitution at G9 and A13 of the ribozyme decreases the catalytic activity (kcat) of the ribozyme by factors of 14 and 20, respectively. The reduction of the reaction rate concomitant with the absence of these 2'-OHs or the 2'-OH of the substrate U7 position can be partially compensated by increasing the Mg2+ concentration above 10 mM. The KMg of the all-RNA ribozyme is 5.3 mM, and the lack of either of the three influential 2'-OHs increases this value by a factor of approximately 3. These and other reaction constants for the ribozyme and the deoxy-substituted analogues have been determined by assuming a three-step mechanism. The data presented here provide the basis for the formulation of a molecular model of ribozyme activity.
The importance of the 2'-hydroxyl group of several guanosine residues for the catalytic efficiency of a hammerhead ribozyme has been investigated. Five ribozymes in which single guanosine residues were substituted with 2'-amino-, 2'-fluoro-, or 2'-deoxyguanosine were chemically synthesized. The comparison of the catalytic activity of the three 2' modifications at a specific position allows conclusions about the functional role of the parent 2'-hydroxyl group. Substitutions of nonconserved nucleotides within the ribozyme caused little alteration in the catalytic activity relative to that obtained with the unmodified ribozyme. In contrast, when either of the guanosines within the single-stranded loop between stem I and stem II of the ribozyme was replaced by 2'-deoxyguanosine or 2'-fluoro-2'-deoxyguanosine, the catalytic activities of the resulting ribozymes were reduced by factors of at least 150. The catalytic activities of the corresponding ribozymes containing 2'-amino-2'-deoxyguanosine substitutions at these positions, however, were both reduced by factors of 15. These effects resulted from decreases in the respective kcat values, whereas variations in the Km values were comparatively small. A different pattern of reactivity of the three 2' modifications was observed at the guanosine immediately 3' to stem II of the ribozyme. Whereas both 2'-deoxyguanosine and 2'-amino-2'-deoxyguanosine at this position showed catalytic activity similar to that of the unmodified ribozyme, the activity of the corresponding 2'-fluoro-2'-deoxyguanosine-containing ribozyme was reduced by a factor of 15. The implications of these substitution-specific reactivities on the functional role of the native 2'-hydroxyl groups are discussed.
The improved synthesis of 2'-fluoro-2'-deoxyadenosine (2'-FA) starting from adenosine is described. This compound was converted to the phosphoramidite and incorporated into a hammerhead ribozyme RNA with the use of automated RNA synthesis techniques. Ribozymes containing 2'-deoxy-adenosine (2'-dA) were prepared in a similar manner. A kinetic rate comparison of the unmodified ribozyme with two ribozymes that had every adenosine replaced with 2'FA or 2'-dA revealed a large decrease in catalytic efficiency (kcat/Km) for the modified ribozymes resulting from a drop in kcat. The kinetic analysis of a number of partially substituted 2'-FA or 2'-dA containing hammerheads revealed that the decrease in activity was not associated with any particular residue but was the result of the accumulation of modified nucleosides within the structure.
We describe a novel DNA and RNA found in the mitochondria of the Varkud-1c natural isolate of Neurospora. The majority of the RNA, termed VSRNA, is an 881 nucleotide single-stranded circular molecule complementary to one strand of a low copy, double-stranded circular DNA, VSDNA. VSRNA combines some features of the human hepatitis delta virus, group I introns, retroelements, and plant viral satellite RNAs. VSRNA synthesized in vitro performs a self-cleavage reaction whose products terminate with a 5' hydroxyl and a 2',3' cyclic phosphate. This reaction may be involved in the natural processing pathway of multimeric VSRNA in vivo. VSRNA lacks a hammer-head structure or substantial sequence similarity to any other self-cleaving RNA, suggesting that the RNA structure involved in cleavage may be different from those in previously characterized catalytic RNAs.
Proteins are not the only catalysts of cellular reactions; there is a growing list of RNA molecules that catalyze RNA cleavage and joining reactions. The chemical mechanisms of RNA-catalyzed reactions are discussed with emphasis on the self-splicing ribosomal RNA precursor of Tetrahymena and the enzymatic activities of its intervening sequence RNA. Wherever appropriate, catalysis by RNA is compared to catalysis by protein enzymes.
We have investigated the in vitro self-splicing of a class II mitochondrial intron. A model pre-mRNA containing intron 5 gamma of the oxi 3 gene of yeast mitochondrial DNA undergoes an efficient intramolecular rearrangement reaction in vitro. This reaction proceeds under conditions distinct from those optimal for self-splicing of class I introns, such as the Tetrahymena nuclear rRNA intron. Intron 5 gamma is excised as a nonlinear RNA indistinguishable from the in vivo excised intron product by gel electrophoresis and primer extension analysis. Studies of the in vitro excised intron product strongly indicate that it is a branched RNA with a circular component joined by a linkage other than a 3'-5' phosphodiester. Two other products, the spliced exons and the broken form of the lariat, were also characterized. These results show that the class II intron products are similar to those of nuclear pre-mRNA splicing.
In the macronuclear rRNA genes of Tetrahymena thermophila, a 413 bp intervening sequence (IVS) interrupts the 26S rRNA-coding region. A restriction fragment of the rDNA containing the IVS and portions of the adjacent rRNA sequences (exons) was inserted downstream from the lac UV5 promoter in a recombinant plasmid. Transcription of this template by purified Escherichia coli RNA polymerase in vitro produced a shortened version of the pre-rRNA, which was then deproteinized. When incubated with monovalent and divalent cations and a guanosine factor, this RNA underwent splicing. The reactions that were characterized included the precise excision of the IVS, attachment of guanosine to the 5' end of the IVS, covalent cyclization of the IVS and ligation of the exons. We conclude that splicing activity is intrinsic to the structure of the RNA, and that enzymes, small nuclear RNAs and folding of the pre-rRNA into an RNP are unnecessary for these reactions. We propose that the IVS portion of the RNA has several enzyme-like properties that enable it to break and reform phosphodiester bonds. The finding of autocatalytic rearrangements of RNA molecules has implications for the mechanism and the evolution of other reactions that involve RNA.
The L-21 ScaI ribozyme catalyzes sequence-specific cleavage of an oligonucleotide substrate. Cleavage is preceded by base pairing of the substrate to the internal guide sequence (IGS) at the 5' end of the ribozyme to form a short RNA duplex (P1). Tertiary interactions between P1 and the catalytic core dock P1 into the active site of the ribozyme. These include interactions between the catalytic core and 2'-hydroxyls of the substrate at nucleotide positions -3u and perhaps -2c. In this study, 2'-hydroxyls of the IGS strand that contribute to P1 recognition by the ribozyme are identified. IGS 2'-hydroxyls (nucleotide positions 22-27) were individually modified to either 2'-deoxy or 2'-methoxynucleotides within full-length semisynthetic L-21 ScaI ribozymes generated using T4 DNA ligase. Thermodynamic and kinetic characterization of the resulting IGS variant ribozymes justify the following conclusions: (i) 2'-Hydroxyls at nucleotide positions G22 and G25 play a critical energetic role in docking P1 into the catalytic core, contributing 2.6 and 2.1 kcal.mol-1, respectively. (ii) The loss of binding energy is manifest primarily as an increase in the rate of dissociation. Because turnover for the wild-type ribozyme is limited by product dissociation, G22 and G25 deoxy variants display up to a 20-fold increase in the multiple-turnover rate at saturating substrate. (iii) IGS tertiary interactions are energetically coupled with the tertiary interactions made to the substrate, consistent with P1 becoming undocked from its binding site in J8/7 upon substitution of either the G22 or G25 2'-hydroxyl. (iv) The G22 deoxy variant loses energetic coupling between guanosine and substrate binding, suggesting that in this variant the P1 helix is also undocked from its binding site in J4/5, the proposed site of guanosine and substrate interaction. Therefore, in combinations with previous studies four P1 2'-hydroxyls are implicated as important for docking. The contributions of the 2'-hydroxyl tertiary interactions are not equivalent and follow the hierarchical order G22 > G25 > -3u > -2c. Because the G22 2'-hydroxyl appears to mediate P1 docking into both J8/7 and J4/5, it may serve as the molecular linchpin for the recognition of P1 by the catalytic core.
In vitro selection can generate functional sequence variants of an RNA structural motif that are useful for comparative analysis.
The technique Is particularly valuable in cases where natural variation is unavailable or non-existent. We report the extension
of this approach to a new extreme—the identification of a 112 nt ribozyme secondary structure Imbedded within a 186 nt RNA.
A pool of 1014 variants of an RNA ligase ribozyme was generated using combinatorial chemical synthesis coupled with combinatorial enzymatic
llgation such that 172 of the 186 relevant positions were partially mutagenized. Active variants of this pool were enriched
using an In vitro selection scheme that retains the sequence variability at positions very close to the ligation junction. Ligases isolated
after four rounds of selection catalyzed self-ligation up to 700 times faster than the starting sequence. Comparative analysis
of the Isolates indicated that when complexed with substrate RNAs the ligase forms a nested, double pseudo-knot secondary
structure with seven stems and several important joining segments. Comparative analys is also suggested the identity of mutations
that account for the Increased activity of the selected ligase variants; designed constructs incorporating combinations of
these changes were more active than any of the Individual ligase isolates.
The Tetrahymena thermophila pre-rRNA contains a 413-nucleotide self-splicing group I intron. This intron has been converted into a sequence-specific endonuclease or ribozyme. A 160-nucleotide portion of the ribozyme consisting of both highly conserved sequence elements (P4 and P6) and nonconserved peripheral extensions (P5abc and P6ab) was synthesized as a separate molecule. Solvent-based Fe(II)-EDTA, a probe that monitors higher-order RNA structure, revealed a protection pattern that was a large subset of that observed in the whole ribozyme. Data from dimethyl sulfate modification and partial digestion with nucleases were also consistent with maintenance of the proper secondary and tertiary structure in the shortened RNA molecule. Thus, this 160-nucleotide molecule (P4-P6 RNA) is an independently folding domain of RNA tertiary structure. A series of mutations and deletions were made within the P4-P6 domain to further dissect its tertiary structure. Fe(II)-EDTA and dimethyl sulfate analysis of these mutants revealed that the domain consists of two substructures, a localized subdomain involving the characteristic adenosine-rich bulge in P5a, and a subdomain-stabilized structure involving long-range interactions. Therefore, like some proteins, the intron RNA is modular, containing a separable domain and subdomain of tertiary structure.
Single-stranded DNA can fold into well-defined sequence-dependent tertiary structures that specifically bind a variety of target molecules, raising the possibility that some folded single-stranded DNAs might exhibit catalytic activities similar to those of ribozymes and protein enzymes. Derivatives of the hammerhead ribozyme that contain a majority of deoxyribonucleotides retain the ability to cleave RNA, and a 'deoxyribozyme' was generated by leaving all essential ribonucleotides of the hammerhead on the RNA 'substrate'. Recently in vitro selection has been used to isolate a DNA sequence that shows Pb(2+)-dependent RNA-cleaving activity. Here we report the isolation by in vitro selection of a small single-stranded DNA that is a Zn2+/Cu(2+)-dependent metalloenzyme. The enzyme catalyses the formation of a new phosphodiester bond by the condensation of the 5'-hydroxyl of one oligodeoxynucleotide and a 3'-phosphorimidazolide on another oligodeoxynucleotide, and shows multiple turnover ligation.
DNA shuffling is a method for in vitro homologous recombination of pools of selected mutant genes by random fragmentation and polymerase chain reaction (PCR) reassembly. Computer simulations called genetic algorithms have demonstrated the importance of iterative homologous recombination for sequence evolution. Oligonucleotide cassette mutagenesis and error-prone PCR are not combinatorial and thus are limited in searching sequence space. We have tested mutagenic DNA shuffling for molecular evolution in a beta-lactamase model system. Three cycles of shuffling and two cycles of backcrossing with wild-type DNA, to eliminate non-essential mutations, were each followed by selection on increasing concentrations of the antibiotic cefotaxime. We report here that selected mutants had a minimum inhibitory concentration of 640 micrograms ml-1, a 32,000-fold increase and 64-fold greater than any published TEM-1 derived enzyme. Cassette mutagenesis and error-prone PCR resulted in only a 16-fold increase.
A processive enzyme binds a polymeric substrate and catalyzes a series of similar chemical reactions along that polymer before releasing the fully modified polymer to solvent. Bovine pancreatic ribonuclease A (RNase A) is a nonprocessive endoribonuclease that binds the bases of adjacent RNA residues in three enzymic subsites: B1, B2, and B3. The B1 subsite binds only to residues having a pyrimidine base, while the B2 subsite prefers adenine and the B3 subsite prefers a purine base. RNase A mutants were created in which all natural amino acids were substituted for Thr45 or Phe120, two residues of the B1 subsite. These pools of mutant enzymes were screened for mutants that catalyze the cleavage of RNA after purine residues. The Ala45 and Gly45 enzymes cleave poly(A), poly(C), and poly(U) efficiently and with 10(3)-10(5)-fold increases in purine/pyrimidine specificity. Thus, substrate binding can be uncoupled from substrate turnover in catalysis by RNase A. In addition, both mutant enzymes cleave poly(A) processively. Our results provide a new paradigm: a processive enzyme has subsites, each specific for a repeating motif within a polymeric substrate. Further, we propose that processive enzymes bind more tightly to motifs that do repeat than to those that do not.
Very complex mutant libraries of the dihydrofolate reductase (DHFR) gene encoded by the Escherichia coli plasmid R67 were created using hypermutagenic PCR with biased deoxynucleotide triphosphate (dNTP) concentrations. Exploiting
the particular stability of the G:T mismatch, the DHFR gene could be enriched in A+T by employing biased deoxypyrimidine triphosphate
concentrations, i.e. [dTTP] < [dCTP]. A sizeable fraction of hypermutants were functional. A combination of [dTTP] < [dCTP]
and [dGTP] < [dATP] biases generated mutations at unexpectedly low frequencies. This could be overcome by the addition of
Mn2+ cations. Overall mutation frequencies of 10% per amplification (range 4–18% per clone) could be attained. All four transitions
and a smaller number of transversions were produced throughout the gene. PCR mutagenesis could be so extensive as to inactivate
all amplified versions of the gene.
THE hypothesis that certain RNA molecules may be able to catalyse RNA
replication is central to current theories of the early evolution of
life1-6. In support of this idea, we describe here an RNA
that synthesizes RNA using the same reaction as that employed by protein
enzymes that catalyse RNA polymerization. In the presence of the
appropriate template RNA and nucleoside triphosphates, the ribozyme
extends an RNA primer by successive addition of up to six
mononucleotides. The added nucleotides are joined to the growing RNA
chain by 3',5'-phosphodiester linkages. The ribozyme shows marked
template fidelity: extension by nucleotides complementary to the
template is up to 1,000 times more efficient than is extension by
mismatched nucleotides.
Group I self-splicing introns catalyze their own excision from precursor RNAs by way of a two-step transesterification reaction.
The catalytic core of these ribozymes is formed by two structural domains. The 2.8-angstrom crystal structure of one of these,
the P4-P6 domain of the Tetrahymena thermophila intron, is described. In the 160-nucleotide domain, a sharp bend allows stacked helices of the conserved core to pack alongside
helices of an adjacent region. Two specific long-range interactions clamp the two halves of the domain together: a two-Mg2+-coordinated adenosine-rich corkscrew plugs into the minor groove of a helix, and a GAAA hairpin loop binds to a conserved
11-nucleotide internal loop. Metal- and ribose-mediated backbone contacts further stabilize the close side-by-side helical
packing. The structure indicates the extent of RNA packing required for the function of large ribozymes, the spliceosome,
and the ribosome.
A population of RNA molecules that catalyze the template-directed ligation of RNA substrates was made to evolve in a continuous
manner in the test tube. A simple serial transfer procedure was used to achieve approximately 300 successive rounds of catalysis
and selective amplification in 52 hours. During this time, the population size was maintained against an overall dilution
of 3 × 10298. Both the catalytic rate and amplification rate of the RNAs improved substantially as a consequence of mutations that accumulated
during the evolution process. Continuous in vitro evolution makes it possible to maintain laboratory “cultures” of catalytic
molecules that can be perpetuated indefinitely.
An in vitro selection procedure was used to develop a DNA enzyme that can be made to cleave almost any targeted RNA substrate under simulated physiological conditions. The enzyme is comprised of a catalytic domain of 15 deoxynucleotides, flanked by two substrate-recognition domains of seven to eight deoxynucleotides each. The RNA substrate is bound through Watson-Crick base pairing and is cleaved at a particular phosphodiester located between an unpaired purine and a paired pyrimidine residue. Despite its small size, the DNA enzyme has a catalytic efficiency (kcat/Km) of approximately 10(9) M-1.min-1 under multiple turnover conditions, exceeding that of any other known nucleic acid enzyme. Its activity is dependent on the presence of Mg2+ ion. By changing the sequence of the substrate-recognition domains, the DNA enzyme can be made to target different RNA substrates. In this study, for example, it was directed to cleave synthetic RNAs corresponding to the start codon region of HIV-1 gag/pol, env, vpr, tat, and nef mRNAs.
The protein enzymes RNA ligase and DNA ligase catalyze the ligation of nucleic acids via an adenosine-5'-5'-pyrophosphate 'capped' RNA or DNA intermediate. The activation of nucleic acid substrates by adenosine 5'-monophosphate (AMP) may be a vestige of 'RNA world' catalysis. AMP-activated ligation seems ideally suited for catalysis by ribozymes (RNA enzymes), because an RNA motif capable of tightly and specifically binding AMP has previously been isolated.
We used in vitro selection and directed evolution to explore the ability of ribozymes to catalyze the template-directed ligation of AMP-activated RNAs. We subjected a pool of 10(15) RNA molecules, each consisting of long random sequences flanking a mutagenized adenosine triphosphate (ATP) aptamer, to ten rounds of in vitro selection, including three rounds involving mutagenic polymerase chain reaction. Selection was for the ligation of an oligonucleotide to the 5'-capped active pool RNA species. Many different ligase ribozymes were isolated; these ribozymes had rates of reaction up to 0.4 ligations per hour, corresponding to rate accelerations of approximately 5 x10(5) over the templated, but otherwise uncatalyzed, background reaction rate. Three characterized ribozymes catalyzed the formation of 3'-5'-phosphodiester bonds and were highly specific for activation by AMP at the ligation site.
The existence of a new class of ligase ribozymes is consistent with the hypothesis that the unusual mechanism of the biological ligases resulted from a conservation of mechanism during an evolutionary replacement of a primordial ribozyme ligase by a more modern protein enzyme. The newly isolated ligase ribozymes may also provide a starting point for the isolation of ribozymes that catalyze the polymerization of AMP-activated oligonucleotides or mononucleotides, which might have been the prebiotic analogs of nucleoside triphosphates.
Background:
RNA and DNA are polymers that lack the diversity of chemical functionalities that make proteins so suited to biological catalysis. All naturally occurring ribozymes (RNA catalysts) that catalyze the formation, transfer and hydrolysis of phosphodiesters require metal-ion cofactors for their catalytic activity. We wished to investigate whether, and to what extent, DNA molecules could catalyze the cleavage (by either hydrolysis or transesterification) of a ribonucleotide phosphodiester in the absence of divalent or higher-valent metal ions or, indeed, any other cofactors.
Results:
We performed in vitro selection and amplification experiments on a library of random-sequence DNA that incorporated a single ribonucleotide, a suitable site for cleavage. Following 12 cycles of selection and amplification, a 'first generation' of DNA enzymes (DNAzymes) cleaved their internal ribonucleotide phosphodiesters at rates approximately 10(7)-fold faster than the spontaneous rate of cleavage of the dinucleotide ApA in the absence of divalent cations. Re-selection from a partially randomized DNA pool yielded 'second generation' DNAzymes that self-cleaved at rates of approximately 0.01 min-1 (a 10(8)-fold rate enhancement over the cleavage rate of ApA). The properties of these selected catalysts were different in key respects from those of metal-utilizing ribozymes and DNAzymes. The catalyzed cleavage took place in the presence of different chelators and ribonuclease inhibitors. Trace-metal analysis of the reaction buffer (containing very high purity reagents) by inductively coupled plasma-optical emission spectrophotometry indicated that divalent or higher-valent metal ions do not mediate catalysis by the DNAzymes.
Conclusions:
Our results indicate that, although ribozymes are sometimes regarded generically to be metalloenzymes, the nucleic acid components of ribozymes may play a substantial role in the overall catalysis. Given that metal cofactors increase the rate of catalysis by ribozymes only approximately 10(2)-10(3)-fold above that of the DNAzyme described in this paper, it is conceivable that substrate positioning, transition-state stabilization or general acid/base catalysis by the nucleic acid components of ribozymes and DNAzymes may contribute significantly to their overall catalytic performance.
Several types of RNA enzymes (ribozymes) have been identified in biological systems and generated in the laboratory. Considering the variety of known RNA enzymes and the similarity of DNA and RNA, it is reasonable to imagine that DNA might be able to function as an enzyme as well. No such DNA enzyme has been found in nature, however. We set out to identify a metal-dependent DNA enzyme using in vitro selection methodology.
Beginning with a population of 10(14) DNAs containing 50 random nucleotides, we carried out five successive rounds of selective amplification, enriching for individuals that best promote the Pb(2+)-dependent cleavage of a target ribonucleoside 3'-O-P bond embedded within an otherwise all-DNA sequence. By the fifth round, the population as a whole carried out this reaction at a rate of 0.2 min-1. Based on the sequence of 20 individuals isolated from this population, we designed a simplified version of the catalytic domain that operates in an intermolecular context with a turnover rate of 1 min-1. This rate is about 10(5)-fold increased compared to the uncatalyzed reaction.
Using in vitro selection techniques, we obtained a DNA enzyme that catalyzes the Pb(2+)-dependent cleavage of an RNA phosphoester in a reaction that proceeds with rapid turnover. The catalytic rate compares favorably to that of known RNA enzymes. We expect that other examples of DNA enzymes will soon be forthcoming.
Previously we demonstrated that DNA can act as an enzyme in the Pb(2+)-dependent cleavage of an RNA phosphoester. This is a facile reaction, with an uncatalyzed rate for a typical RNA phosphoester of approximately 10(-4) min-1 in the presence of 1 mM Pb(OAc)2 at pH 7.0 and 23 degrees C. The Mg(2+)-dependent reaction is more difficult, with an uncatalyzed rate of approximately 10(-7) min-1 under comparable conditions. Mg(2+)-dependent cleavage has special relevance to biology because it is compatible with intracellular conditions. Using in vitro selection, we sought to develop a family of phosphoester-cleaving DNA enzymes that operate in the presence of various divalent metals, focusing particularly on the Mg(2+)-dependent reaction.
We generated a population of > 10(13) DNAs containing 40 random nucleotides and carried out repeated rounds of selective amplification, enriching for molecules that cleave a target RNA phosphoester in the presence of 1 mM Mg2+, Mn2+, Zn2+ or Pb2+. Examination of individual clones from the Mg2+ lineage after the sixth round revealed a catalytic motif comprised of a three-stem junction. This motif was partially randomized and subjected to seven additional rounds of selective amplification, yielding catalysts with a rate of 0.01 min-1. The optimized DNA catalyst was divided into separate substrate and enzyme domains and shown to have a similar level of activity under multiple turnover conditions.
We have generated a Mg(2+)-dependent DNA enzyme that cleaves a target RNA phosphoester with a catalytic rate approximately 10(5)-fold greater than that of the uncatalyzed reaction. This activity is compatible with intracellular conditions, raising the possibility that DNA enzymes might be made to operate in vivo.
Helix packing is critical for RNA tertiary structure formation, although the rules for helix-helix association within structured RNAs are largely unknown. Docking of the substrate helix into the active site of the Tetrahymena group I ribozyme provides a model system to study this question. Using a novel chemogenetic method to analyze RNA structure in atomic detail, we report that complementary sets of noncanonical base pairs (a G.U wobble pair and two consecutively stacked sheared A.A pairs) create an RNA helix packing motif that is essential for 5'-splice site selection in the group I intron. This is likely to be a general motif for helix-helix interaction within the tertiary structures of many large RNAs.