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Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach
Abstract
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... Over the years, the phosphoramidites 8 have been subjected to minor structural modifications in order to improve the stability of phosphoramidite monomers to the conditions used for solid-phase DNA synthesis. These modifications include the replacement of the original dimethylamino function with other N,Ndialkylamino or N,N-cycloalkylamino groups and replacement of the methoxy P(III) protection with a variety of functionalized alkoxy groups (Beaucage and Iyer, 1992;Iyer and Beaucage, 1999) often at the expense of phosphoramidite reactivity. Out of all those proposed phosphoramidite modifications, the most popular deoxyribonucleoside phosphoramidites that have been used since the mid-1980s are the phosphoramidites 11 (Figure 1; Sinha et al., 1984). ...
... However, for specific applications, nonaqueous oxidizing reagents may advantageously offer an alternative to aqueous iodine for the oxidation of oligodeoxyribonucleoside phosphite triesters. These reagents (Beaucage and Iyer, 1992;Uzagare et al., 2003) include m-chloroperbenzoic acid, iodobenzene diacetate and tetra-n-butylammonium periodate, tert-butyl hydroperoxide and various hydroperoxides, dimethyldioxirane and ethyl(methyl)dioxirane, (1S)-( þ )-(10-camphorsulfonyl)oxaziridine, and N-bromosuccinimide-dimethyl sulfoxide (DMSO). Out of these oxidants, an aqueous solution of iodine in pyridine, tert-butyl hydroperoxide and (1S)-( þ )-(10-camphorsulfonyl)oxaziridine are most commonly used in the solid-phase synthesis of native and modified DNA sequences. ...
Synthetic DNA and RNA sequences have been and are still broadly used in the development of a plethora of therapeutic, diagnostic and genomic applications. This report provides updates on improvements made to the phosphoramidite approach to solid-phase synthesis of native and modified nucleic acid sequences with the objective of increasing the quality of those synthetic nucleic acid sequences in terms of purity to ensure the safety and efficacy of potential nucleic acid-based drugs for the treatment of human diseases. The use of deoxyribonucleoside H-phosphonate monomers, as an alternative to phosphoramidite monomers, is highlighted to demonstrate the versatility of the H-phosphonate approach to the solid-phase synthesis of modified oligonucleotides. Furthermore, the solid-phase synthesis of RNA sequences is substantially revised with a particular emphasis on the selection of 2′-hydroxyl protecting groups for ribonucleosides and their phosphoramidite derivatives. Updates have been made to better address issues associated with the manufacture of RNA sequences, particularly in the context of drug discovery and cellular/tissue delivery for potential therapeutic applications.
... The stage at which the strands or DNA molecules that encode the data are produced (those strands called input strands, or variants). In order to produce strands with acceptable error rates, in a high throughput manner, the length of the strands is typically limited to no more than 250 nucleotides [2]. The second part is a storage container with compartments. ...
... • Description: For every position between 0 (the 1 For example: the deletion rate in Fig. 1 is 24/(25 × 27), which is calculated to be the ratio between the number of red squares (24) and the product of the number of rows (25) with the variant length (27). 2 The expected number of base x in the reads is calculated as the sum of the products of the number of base x in each of the design variants, and the number of reads matched to it. ...
Motivation
Recent years have seen a growing number and a broadening scope of studies using synthetic oligo libraries for a range of applications in synthetic biology. As experiments are growing by numbers and complexity, analysis tools can facilitate quality control and help in assessment and inference.
Results
We present a novel analysis tool, called SOLQC , which enables fast and comprehensive analysis of synthetic oligo libraries, based on NGS analysis performed by the user. SOLQC provides statistical information such as the distribution of variant representation, different error rates and their dependence on sequence or library properties. SOLQC produces graphical descriptions of the analysis results. The results are reported in a flexible report format. We demonstrate SOLQC by analyzing literature libraries. We also discuss the potential benefits and relevance of the different components of the analysis.
Availability
https://app.gitbook.com/@yoav-orlev/s/solqc/
... Nowadays, coating silica particles with alkoxysilanes by the formation of strong covalent Si−O−Si bonds to such surfaces 23,24 leads to a large number of new interesting applications. 25,26 One of these are solid supports [e.g., controlled pore glass (CPG) silicas, 27 as well as polymeric and hybrid ones 28,29 ], which allow nucleic acids to be synthesized on solid phase but with solution interaction. Using CPG gives a larger surface for growing the oligonucleotide chain. ...
... The nucleic acid was attached to the support by phosphate linkage using phosphoramidite chemistry. 28 Polypropylene 29 and polystyrene 37−39 are currently the most commonly used as supports for permanent anchored oligonucleotides. However, a more and more popular functional surface used in phosphoramidite chemistry is functional glass surfaces because the swelling of the support (observed for polymers) is eliminated. ...
In this paper, the Pt-catalyzed hydrosilylation of hydroxyl ethers is described. Various bifunctional alkoxysilanes were obtained and applied in O-silylation of free hydroxyl groups on the silica surface. These modified solid materials have been used as excellent supports for linking synthetic nucleic acids. Nucleic acids permanently attached to the solid surface were tested in hybridization with complementary fluorescence-labeled sequences. Detection of nucleic acids anchored to the solid support was performed by fluorescence microscopy after hybridization.
... Over the years, the phosphoramidites 8 have been subjected to minor structural modifications in order to improve the stability of phosphoramidite monomers to the conditions used for solid-phase DNA synthesis. These modifications include the replacement of the original dimethylamino function with other N,Ndialkylamino or N,N-cycloalkylamino groups and replacement of the methoxy P(III) protection with a variety of functionalized alkoxy groups (Beaucage and Iyer, 1992;Iyer and Beaucage, 1999) often at the expense of phosphoramidite reactivity. Out of all those proposed phosphoramidite modifications, the most popular deoxyribonucleoside phosphoramidites that have been used since the mid-1980s are the phosphoramidites 11 (Figure 1; Sinha et al., 1984). ...
... However, for specific applications, nonaqueous oxidizing reagents may advantageously offer an alternative to aqueous iodine for the oxidation of oligodeoxyribonucleoside phosphite triesters. These reagents (Beaucage and Iyer, 1992;Uzagare et al., 2003) include m-chloroperbenzoic acid, iodobenzene diacetate and tetra-n-butylammonium periodate, tert-butyl hydroperoxide and various hydroperoxides, dimethyldioxirane and ethyl(methyl)dioxirane, (1S)-( þ )-(10-camphorsulfonyl)oxaziridine, and N-bromosuccinimide-dimethyl sulfoxide (DMSO). Out of these oxidants, an aqueous solution of iodine in pyridine, tert-butyl hydroperoxide and (1S)-( þ )-(10-camphorsulfonyl)oxaziridine are most commonly used in the solid-phase synthesis of native and modified DNA sequences. ...
Abstract Synthetic DNA and RNA sequences have been and are still heavily used in the development of a plethora of therapeutic, diagnostic, and genomic applications. This report describes the details of the phosphoramidite approach to the solid-phase synthesis of DNA sequences including optimization of the critical steps that have been made over the years to improve the coupling kinetics and efficiency of deoxyribonucleoside phosphoramidite monomers. The use of deoxyribonucleoside H-phosphonate monomers, as an alternative to phosphoramidite monomers, is presented in sufficient detail to highlight the versatility of the H-phosphonate approach to the synthesis of native and modified oligonucleotides despite its inherent limitations. The solid-phase synthesis of RNA sequences is also reviewed with a particular emphasis on the selection of 2′-hydroxyl protecting groups for ribonucleosides and their phosphoramidite derivatives to underscore this critical issue in the synthesis of RNA sequences. The in situ synthesis of DNA sequences on microarrays using photolithographic, inkjet printing, electrochemical, and microfluidic processes is reported in the context of diagnostic, drug discovery, and gene assembly applications. The usefulness of DNA microarrays in the fabrication of RNA microarrays is demonstrated while the difficulties of in situ RNA synthesis on glass surfaces remain to be overcome.
... Phosphoramidite chemistry allows the facile synthesis of natural nucleic acids as well as oligomers containing various unnatural monomers [1]. By using this technology, xenonucleic acids (XNAs), which are DNA analogs, have also been developed to improve the performance of nucleic acid-based biological tools and to elucidate why D-ribose is the naturally selected scaffold for genetic carriers [2][3][4][5][6][7][8]. ...
... However, at first the yield of PS analog was low due to the inefficiency of the sulfurization reaction 23 compared to the usual oxidation reaction. 24,25 We were able to increase the yield using a different solvent. 26 The oligomers were tested in an HIV assay and the PS oligomers showed excellent inhibition against the rev gene, but none with the PM oligos or the PO oligo controls. ...
In the search for novel therapeutics, antisense oligonucleotide (ASO) analogs have been a major focus of research for over 40 years. They use the antisense strategy, namely they have a nucleic acid base sequence that is complementary to a portion of a specific mRNA that is produced in the cell, or to a viral RNA, in order to selectively inhibit gene expression. Oligonucleotides need to be chemically modified to stabilize them against hydrolysis by endogenous nucleases. Until now several phosphorothioate (PS) oligonucleotide analogs have been approved by the FDA for human use. This article seeks to provide a history of this subject to date.
... All oligos were synthesized using an automated DNA synthesizer and purified by HPLC and ethanol precipitation (36). ...
Mitochondrial transcription factor A (TFAM) plays a critical role in mitochondrial transcription initiation and mitochondrial DNA (mtDNA) packaging. Both functions require DNA binding, but in one case TFAM must recognize a specific promoter sequence, while packaging requires coating of mtDNA by association with non sequence-specific regions. The mechanisms by which TFAM achieves both sequence-specific and non sequence-specific recognition have not yet been determined. Existing crystal structures of TFAM bound to DNA allowed us to identify two guanine-specific interactions that are established between TFAM and the bound DNA. These interactions are observed when TFAM is bound to both specific promoter sequences and non-sequence specific DNA. These interactions are established with two guanine bases separated by 10 random nucleotides (GN10G). Our biochemical results demonstrate that the GN10G consensus is essential for transcriptional initiation and contributes to facilitating TFAM binding to DNA substrates. Furthermore, we report a crystal structure of TFAM in complex with a non sequence-specific sequence containing a GN10G consensus. The structure reveals a unique arrangement in which TFAM bridges two DNA substrates while maintaining the GN10G interactions. We propose that the GN10G consensus is key to facilitate the interaction of TFAM with DNA.
... The chainextending cycles are repeated until the synthesis of the last oligonucleotide is completed. The traditional phosphoramidite method of the synthesis is used in this strategy [5] except that the acid-labile 4,4'-dimethoxitritil group is replaced with the photolabile protecting group. ...
The application of array-based oligonucleotides in biological studies is described. These oligonu-cleotides are mainly used to design large libraries of various nucleotide sequences, which are applied to study protein-nucleic acid interactions, splicing, transcription, translation, and other regulatory processes in mam-malian, yeast, and bacterial systems. The application of gene libraries generated by array-based nucleotides along with advanced methods of the combination of DNA duplexes will make it possible to obtain complex genetic designs for synthetic biology.
... As mentioned in the Conspectus, these polymers are synthesized by solid-phase iterative phosphoramidite chemistry; a technique that has been historically developped for the chemical-synthesis of DNA. 25 Still, the phosphoramidite approach is not restricted to nucleoside monomers and a wide variety of non-natural building blocks can also be employed in this technique. 26,27 In fact, when all building blocks are nonnatural, phosphoramidite chemistry is no longer a biochemical technique but simply a polymer chemistry tool. ...
ConspectusIn biological systems, the storage and transfer of genetic information rely on sequence-controlled nucleic acids such as DNA and RNA. It has been realized for quite some time that this property is not only crucial for life but could also be very useful in human applications. For instance, DNA has been actively investigated as a digital storage medium over the past decade. Indeed, the "hard-disk of life" is an obvious choice and a highly optimized material for storing data. Through decades of nucleic acids research, technological tools for parallel synthesis and sequencing of DNA have been readily available. Consequently, it has already been demonstrated that different types of documents (e.g., texts, images, videos, and industrial data) can be stored in chemically synthesized DNA libraries. However, DNA is subject to biological constraints, and its molecular structure cannot be easily varied to match technological needs. In fact, DNA is not the only macromolecule that enables data storage. In recent years, it has been demonstrated that a wide variety of synthetic polymers can also be used for such a purpose. Indeed, modern polymer synthesis allows the preparation of synthetic macromolecules with precisely controlled monomer sequences. Altogether, about a dozens of synthetic digital polymers have already been described, and many more can be foreseen. Among them, sequence-defined poly(phosphodiester)s are one of the most promising options. These polymers are prepared by stepwise phosphoramidite chemistry like chemically synthesized oligonucleotides. However, they are constructed with non-natural building blocks and therefore share almost no structural characteristics with nucleic acids, except phosphate repeat units. Still, they contain readable digital messages that can be deciphered by nanopore sequencing or mass spectrometry sequencing. In this Account, we describe our recent research efforts in synthesizing and sequencing optimal abiological digital poly(phosphodiester)s. A major advantage of these polymers over DNA is that their molecular structure can easily be varied to tune their properties. During the last 5 years, we have engineered the molecular structure of these polymers to adjust crucial parameters such as the storage density, storage capacity, erasability, and readability. Consequently, high-capacity PPDE chains, containing hundreds of bits per chains, can now be synthesized and efficiently sequenced using a routine mass spectrometer. Furthermore, sequencing data can be automatically decrypted with the help of decoding software. This new type of coded matter can also be edited using practical physical triggers such as light and organized in space by programmed self-assembly. All of these recent improvements are summarized and discussed herein.
... The first is the DNA synthesis which produces the oligonucleotides, also called strands, that encode the data. In order to produce strands with acceptable error rates, in a high throughput manner, the length of the strands is typically limited to no more than 250 nucleotides [5]. The second part is a storage container with compartments which stores the DNA strands, however without order. ...
In the trace reconstruction problem a length- n string x yields a collection of noisy copies, called traces , y 1 , …, y t where each y i is independently obtained from x by passing through a deletion channel , which deletes every symbol with some fixed probability. The main goal under this paradigm is to determine the required minimum number of i.i.d traces in order to reconstruct x with high probability. The trace reconstruction problem can be extended to the model where each trace is a result of x passing through a deletion-insertion-substitution channel , which introduces also insertions and substitutions. Motivated by the storage channel of DNA, this work is focused on another variation of the trace reconstruction problem, which is referred by the DNA reconstruction problem . A DNA reconstruction algorithm is a mapping which receives t traces y 1 , …, y t as an input and produces , an estimation of x . The goal in the DNA reconstruction problem is to minimize the edit distance between the original string and the algorithm’s estimation. For the deletion channel case, the problem is referred by the deletion DNA reconstruction problem and the goal is to minimize the Levenshtein distance .
In this work, we present several new algorithms for these reconstruction problems. Our algorithms look globally on the entire sequence of the traces and use dynamic programming algorithms, which are used for the shortest common supersequence and the longest common subsequence problems, in order to decode the original sequence. Our algorithms do not require any limitations on the input and the number of traces, and more than that, they perform well even for error probabilities as high as 0.27. The algorithms have been tested on simulated data as well as on data from previous DNA experiments and are shown to outperform all previous algorithms.
... However, at first the yield of PS analog was low due to the inefficiency of the sulfurization reaction 23 compared to the usual oxidation reaction. 24,25 We were able to increase the yield using a different solvent. 26 The oligomers were tested in an HIV assay and the PS oligomers showed excellent inhibition against the rev gene, but none with the PM oligos or the PO oligo controls. ...
In the search for novel therapeutics, antisense oligonucleotide (ASO) analogs have been a major focus of research for over 40 years. They use the antisense strategy, namely they have a nucleic acid base sequence that is complementary to a portion of a specific mRNA that is produced in the cell, or to a viral RNA, in order to selectively inhibit gene expression. Oligonucleotides need to be chemically modified to stabilize them against hydrolysis by endogenous nucleases. Until now several phosphorothioate (PS) oligonucleotide analogs have been approved by the FDA for human use. This article seeks to provide a history of this subject to date.
... So far, aptamers have been developed for a wide variety of targets, such as small molecules [27][28][29], ions [30,31], proteins [32,33], and cells [34][35][36]. The ability of aptamers to be reproduced in large scales [37] and with little variation [38], and modification easily with reporter molecules [39] makes aptamers ideal as a molecular recognition element in sensor applications. To determine the metal ions in an aqueous medium, DNA aptamers were designed such as G-quadruplex probes [40] for Pb 2þ , cytosine-cytosine mismatch [41] for Ag þ , thymine-rich and guanine-rich probes [42] for Cd 2þ , and thymine-thymine mismatch [43] for Hg 2þ . ...
Heavy metal ion pollution is a severe problem in environmental protection and especially in human health due to their bioaccumulation in organisms. Mercury (II) (Hg²⁺), even at low concentrations, can lead to DNA damage and give permanent harm to the central nervous system by easily passing through biological membranes. Therefore, sensitive detection and monitoring of Hg²⁺ is of particular interest with significant specificity. In this review, aptamer-based strategies in combination with nanostructures as well as several other strategies to solve addressed problems in sensor development for Hg²⁺ are discussed in detail. In particular, the analytical performance of different aptamer and oligonucleotide-based strategies using different signal improvement approaches based on nanoparticles were compared within each strategy and in between. Although quite a number of the suggested methodologies analyzed in this review fulfills the standard requirements, further development is still needed on real sample analysis and analytical performance parameters.
... The starting modified oligonucleotides (ONs) and DNA can be synthesized either chemically or enzymatically (Scheme 1). The automated chemical synthesis is performed on solid support using modified nucleoside phosphoramidites [7]. Unfortunately, many reactive groups (electrophiles, nucleophiles etc.) are not compatible with the phosphoramidite protocol and/or deprotection with ammonia, and therefore often they must be masked or protected. ...
Modification of DNA with reactive groups and their post-synthetic transformations are useful for labelling, imaging, bioconjugations and cross-linking with other (bio)molecules. This review summarizes the recent progress in this field and covers transformations of oxo groups, cycloadditions, conjugate additions, alkylations, cross-couplings and other reactions. Examples of applications are given and the practicability and scope of the reactions are discussed.
... Also, while other researches compared in Fig. 1B used library of more than thousands copies of each oligonucleotide sequences, we achieved an empirical information capacity of more than 2bits per character within an oligonucleotide library comprising hundreds of copies for each sequence. The degenerative portion of the encoded sequence is incorporated by mixing the DNA phosphoramidites during the synthetic procedure 12 and generating variants of the corresponding combinations of A, C, G and T (Fig. 1C,D). Ideally, for column-based 12 and inkjet-based [13][14][15] oligonucleotide synthesis, degenerate bases can be added without extra cost because the total amount of phosphoramidites used is the same(Supplementary Note). ...
DNA-based data storage has emerged as a promising method to satisfy the exponentially increasing demand for information storage. However, practical implementation of DNA-based data storage remains a challenge because of the high cost of data writing through DNA synthesis. Here, we propose the use of degenerate bases as encoding characters in addition to A, C, G, and T, which augments the amount of data that can be stored per length of DNA sequence designed (information capacity) and lowering the amount of DNA synthesis per storing unit data. Using the proposed method, we experimentally achieved an information capacity of 3.37 bits/character. The demonstrated information capacity is more than twice when compared to the highest information capacity previously achieved. The proposed method can be integrated with synthetic technologies in the future to reduce the cost of DNA-based data storage by 50%.
... Phosphoramidites were first proposed in the early 1980′s [27] and have since come to dominate oligonucleotide synthesis [28]. Phosphoramidites are generally highly reactive towards nucleophiles in the presence of even weak acids [29]. Previous to their introduction, chemical DNA synthesis had been conducted using H-phosphonate [30][31][32][33], phosphodiester [34], phosphotriester [35,36] and phosphite triester [37,38] synthetic routes. ...
RNA is often considered as being the vector for the transmission of genetic information from DNA to the protein synthesis machinery. However, besides translation RNA participates in a broad variety of fundamental biological roles such as gene expression and regulation, protein synthesis, and even catalysis of chemical reactions. This variety of function combined with intricate three-dimensional structures and the discovery of over 100 chemical modifications in natural RNAs require chemical methods for the modification of RNAs in order to investigate their mechanism, location, and exact biological roles. In addition, numerous RNA-based tools such as ribozymes, aptamers, or therapeutic oligonucleotides require the presence of additional chemical functionalities to strengthen the nucleosidic backbone against degradation or enhance the desired catalytic or binding properties. Herein, the two main methods for the chemical modification of RNA are presented: solid-phase synthesis using phosphoramidite precursors and the enzymatic polymerization of nucleoside triphosphates. The different synthetic and biochemical steps required for each method are carefully described and recent examples of practical applications based on these two methods are discussed.
... First, N,Ndimethyltryptamine (DMT) depro tection of the monomer occurs, allowing the connec tion of the next monomer by phosphoramidite coupling, followed by oxidation of the phosphite to a phosphate. Optimization of this method allows each threestep cycle to be completed within a few minutes 128 . Lutz et al. used this approach to synthesize a polymer with a controlled sequence from two monomers containing either a propyl moiety (representing 0) or a 2,2dimethylpropyl moiety (representing 1) 129 . ...
Defined-sequence polymers have great potential as durable and high-density data-storage media. DNA already fulfils this role in nature, using the sequence of its four nucleobases to store genetic information. Synthetic DNA can be used to store binary codes, and it is both more durable and can store information at a much higher density than conventional silicon-based storage systems. Other defined-sequence synthetic polymers have properties that make them even more suitable for data storage, at least in principle, assuming that complete control over their composition, that is, their monomer sequence, can be achieved. This Review addresses the current status of data storage in DNA, proteins and synthetic polymers, with the objective to overcome the problems of current data storage technology.
... Parallel to advances in isotope enrichment of proteins, specific labeling strategies for nucleic acids were established during the past decades. Deoxynucleotides with specific labels can be synthesized chemically by phosphoramidite-based solid phase synthesis 110,111 and specific labels can be incorporated with ease 112,113 . Zimmer and Crothers first demonstrated enzymatic synthesis of labeled DNA where they designed self-priming hairpin ssDNA with modified 3′ terminal ribonucleotide 114 . ...
NMR spectroscopy has become an indispensable tool for high-resolution structure determination of biomolecules at physiological conditions both in solutions and solids. Currently, NMR is routinely used to study the structure and dynamics of high molecular weight biomolecules in sizes ranging up to ~ 50–100 kDa and to evaluate complexes as large as 500–1 MDa. The latest advances in spectrometer technology, methodologies and advents in newer and highly innovative NMR active isotope-labeling strategies now enable us to overcome an earlier speculated size barrier of ~ 20 kDa for de novo structure determination. Of these, developments in NMR active isotope-labeling strategies are of great significance as they allow reduction in spectral crowding and yield selective spin correlations. Moreover, NMR isotope enrichment schemes permit exploitation of heteronuclear magnetization transfer pathways for enhanced sensitivity and selectivity. Functionally relevant sites or domains in very large complexes can also be selectively evaluated by specific labeling strategies in which other regions are masked. Further, labeling schemes can be effectively used to favourably overcome deleterious relaxation effects. Recently evolved labeling strategies include uniform labeling, perdeuteration, specific labeling of an amino acid or a side chain, selective deuteration or protonation, segmental labeling and biosynthesis of biomolecules in various organisms, cell lines and cell-free systems. The present review is aimed at introducing various NMR isotope labeling strategies and discusses their impact in widening the scope of biomolecular NMR spectroscopy driven structural biology.
... To the best of our knowledge, the synthesis of phosphoramidite 3 (Figure 2) has not been previously reported. (51,63,64). This monomer was used to automatically synthesize the modified TBA containing W ( Figure 1B and Supplementary Table ST-1) using the standard phosphoramidite method (51,65). ...
Thrombin-binding aptamer (TBA) is a DNA 15-mer of sequence 5'-GGT TGG TGT GGT TGG-3' that folds into a G-quadruplex structure linked by two T-T loops located on one side and a T-G-T loop on the other. These loops are critical for post-SELEX modification to improve TBA target affinity. With this goal in mind we synthesized a T analog, 5-(indolyl-3-acetyl-3-amino-1-propenyl)-2'-deoxyuridine (W) to substitute one T or a pair of Ts. Subsequently, the affinity for each analog was determined by biolayer interferometry. An aptamer with W at position 4 exhibited about 3-fold increased binding affinity, and replacing both T4 and T12 with W afforded an almost 10-fold enhancement compared to native TBA. To better understand the role of the substituent's aromatic moiety, an aptamer with 5-(methyl-3-acetyl-3-amino-1-propenyl)-2'-deoxyuridine (K; W without the indole moiety) in place of T4 was also synthesized. This K4 aptamer was found to improve affinity 7-fold relative to native TBA. Crystal structures of aptamers with T4 replaced by either W or K bound to thrombin provide insight into the origins of the increased affinities. Our work demonstrates that facile chemical modification of a simple DNA aptamer can be used to significantly improve its binding affinity for a well-established pharmacological target protein.
... Solid phase synthesis is a fully chemical method for the production of oligonucleotides starting from phosphoramidites. Developed in the 1980s, the method is routinely used to produce large quantities (up to gram scale) of 2′-deoxyoligonculeotides (DNA) for numerous biochemical and medical applications (Fig. 1a) (Beaucage and Iyer 1992;Reese 2005). The general structural formula of the RNA phosphoramidites and the 2′-protecting groups are shown in Fig. 1b. ...
Nucleic acids play key roles in most biological processes, either in isolation or in complex with proteins. Often they are difficult targets for structural studies, due to their dynamic behavior and high molecular weight. Solid-state nuclear magnetic resonance spectroscopy (ssNMR) provides a unique opportunity to study large biomolecules in a non-crystalline state at atomic resolution. Application of ssNMR to RNA, however, is still at an early stage of development and presents considerable challenges due to broad resonances and poor dispersion. Isotope labeling, either as nucleotide-specific, atom-specific or segmental labeling, can resolve resonance overlaps and reduce the line width, thus allowing ssNMR studies of RNA domains as part of large biomolecules or complexes. In this review we discuss the methods for RNA production and purification as well as numerous approaches for isotope labeling of RNA. Furthermore, we give a few examples that emphasize the instrumental role of isotope labeling and ssNMR for studying RNA as part of large ribonucleoprotein complexes.
... Despite the difficulties of this approach, short sequences of DNA with a known sequences were employed for structural characterization X-ray crystallography and NMR [6,7], studies of drugs-binding (distamycins [8], daunomycins [9] etc.), and as antisense gene down-regulators [10]. In the '80s, following the development of the intuition of Beaucage and Carruthers [11], a new strategy of synthesis based on the high reactivity of P(III) was developed [12] and found a widespread application toward the automated synthesis starting from the 3'-terminal nucleoside attached to a solid support normally controlled pore glass (CPG). Since then little has changed, but as chemists, we were challenged with the poor performance of exogenous DNA. ...
In the last 30 years oligonucleotides i.e., relatively short polymers (usually 12-24 units long) based on DNA structure, have found a widespread use in biochemical studies and as biochemical probes and chemotherapeutic agents for the downregulation of genetic expression or for exon skipping. Here we present a short review of studies from our laboratories on the synthesis and applications of different kind of conjugates to address some of these techniques. Preparation of conjugates with small alkyl groups, intercalators, fluorescent oligothiophenes, and lipophilic bile-acids will be discussed.
... RNA, in most cases, consists one strand of nucleic acids, using uracil (U) instead of thymine (T) in its nucleobase pairing [20]. Besides the biosynthesis of DNA/RNA in nature, oligonucleotide synthesis is based on phosphoramidite chemistry, which was developed in the 1980s and later combined with solid-phase supports and automation [21,22] (Figure 1). However, the length of synthetic oligonucleotide is limited by the coupling efficiency and the following purification steps, which resulted in a typical length of synthetic single-stranded DNA/RNA of around 70~100 bases, with low error rates [23]. ...
Nucleobase interactions play a fundamental role in biological functions, including transcription and translation. Natural nucleic acids like DNA are also widely implemented in material realm such as DNA guided self-assembly of nanomaterials. Inspired by that, polymer chemists have contributed phenomenal endeavors to mimic both the structures and functions of natural nucleic acids in synthetic polymers. Similar sequence-dependent responses were observed and employed in the self-assembly of these nucleobase-containing polymers. Here, the structures, synthetic approaches, and applications of nucleobase-containing polymers are highlighted and a brief look is taken at the future development of these polymers.
... The building blocks are gradually coupled to the oligonucleotide chain. The number of errors generated sets the practical limits of the length of the chain being synthesized (Beaucage and Iyer 1992). The products of oligonucleotide synthesis are isolated by high-performance liquid chromatography (HPLC) to obtain the desired oligonucleotides in high purity. ...
We present a new theoretical technical solution application of nanopore sequencing (Oxford Nanopore Technologies, https://www. nanoporetech.com/), which has primarily been used to read molecules. We describe how to apply nanopore sequencing for the production of a new type of device synthesing of molecules according to our requirements of a chosen database that has been prepared in advance. This technology can implement the programmed synthesis of long DNA strings, ideally entire chromosomes. We propose a device using nanopores and a system of nanotubes that can synthesize molecules using individual nucleotides, existing single-stranded DNA (ssDNA) ligase or modified forms of ssDNA ligase and the energy provided by ATP. The second strand is synthesized by Taq polymerase, which is located below the nanopore. The synthesis of long strings is performed using many nanopores positioned linearly. The strings are collected in wells below the nanopore, after which the strings are ligated to form a long single string (or to form part or all of a chromosome) by double-stranded DNA (dsDNA) ligase. There are many options for successful implementation, particularly in the field of genetic engineering.
... Oligonucleotide solid phase synthesis of modified oligonucleotides was performed in accordance with the standard protocol [32][33][34]. After the synthesis the oligomers were deprotected and cleaved from the solid support by treatment with ammoniasaturated methanol (approx. ...
Herein, we describe synthesis of novel acyclic dinucleotide analogues connected via triazole linkage in CuAAC reaction. Synthesis pathway starting from previously obtained building blocks containing alkyne or azide functional group is described. Further functionalization and application of dinucleotide analogues in DNA phosphoramidite solid-phase synthesis is also explained. Additionally, we have examined the influence of novel modifications on DNA duplex thermodynamic stability.
... In this study, we show a characterization of a hyperthermophilic esterase from Pyrococcus furiosus. The region of the gene AFN03900.1 (amino acids 160-404), predicted as an esterase was synthesized (GenOne Biotechnologies-Rio de Janeiro, Brazil) by a solid-phase synthesis using phosphoramidite method [12] ( Figure S1). The gene was synthesized based on the nucleotide sequence, delivered in the vector pBluescript II SK ( Figure S2), and digested with the NheI and BamHI restriction enzymes and then inserted into the expression vector pET-28a. ...
Enzymes isolated from extremophiles often
exhibit superior performance and potential industrial
applications. There are several advantages performing
biocatalysis at elevated temperatures, including enhanced
reaction rates, increased substrate solubility and decreased
risks of contamination. Furthermore, thermophilic enzymes
usually exhibit high resistance against many organic solvents
and detergents, and are also more resistant to proteolytic
attack. In this study, we subcloned and characterized
an esterase from the hyperthermophilic archaeon Pyrococcus
furiosus (Pf_Est) that exhibits optimal activity
around 80 �C using naphthol-derived substrates and p-nitrophenyl
palmitate (pNPP). According to the circular
dichroism spectra, the secondary structure of P. furiosus
esterase, which is predominantly formed by a b-sheet
structure, is very stable, even after incubation at 120�C. We
performed SAXS to determine the low-resolution structure
of Pf_Est, which is monomeric in solution at 80 �C and has
a molecular weight of 28 kDa. The Km and Vmax values for
this esterase acting on pNPP were 0.53 mmol/L and
6.5 9 10-3 U, respectively. Pf_Est was most active in the
immiscible solvents and retained more than 50 % in miscible
solvents. Moreover, Pf_Est possesses transesterification
capacity, presenting better results when isobutanol was
used as an acyl acceptor (2.69 ± 0.14 9 10-2 lmol/
min mg) and the highest hydrolytic activity toward olive
oil among different types of oils testes in this study. Collectively,
these biophysical and catalytic properties are of
interest for several biotechnological applications that
require harsh conditions, including high temperature and
the presence of organic solvents.
... Oligonucleotides are short sequences of DNA and/or RNA that have unique properties. They can be synthesized up to 160-200 bp 4 . These compounds are commonly used as starters in polymerase chain reaction, in gene delivery and as drugs for some diseases such as a virus-associated illness, AIDS, Alzheimer, cardiovascular disorder, and cancer [5][6][7]. ...
The main aim of the present investigation was to determine retention behavior and interactions of oligonucleotides with alkylamide stationary phase. Five oligonucleotides, differing in the sequence, were tested. Changes in the composition of the mobile phase, i.e. pH (5.0−7.0) and buffer concentration (30−75 mM) were investigated in detail. In addition, the hydrophobic and electrostatic parameters were measured for the different pH’s and salt concentrations. The theoretical model of interactions between individual elements of the separation system, (e.g. solute, stationary phase, and mobile phase) based on zeta potential measurements, hydrophobic and electrostatic parameters calculation, and molecular modeling, has been considered.
... In this figure, the base is guanine. described, including acetyl (ac), benzoyl (bz), dimethylformamidinyl (dmf), isobutyryl (ibu), phenoxyacetyl (pac), isopropylphenoxyacetyl (iPr-pac) and t-butyl-phenoxyacetyl (tac) [11] (Figure 3). ...
The reaction of 2'-deoxynucleoside phosphoramidites with water is an important degradation reaction that limits the lifetimes of reagents used for chemical deoxyoligonucleotide synthesis. The hydrolysis of nucleoside phosphoramidites in solution has therefore been investigated. The degree of degradation depends not only on the presence of water but also on the specific nucleoside, 2'-deoxyguanosine (dG) being especially susceptible. Additionally, the nature of the group protecting the exocyclic amine on the nucleoside base strongly influences the rate of hydrolysis. For dG, the degradation is second order in phosphoramidite concentration, indicating autocatalysis of the hydrolysis reaction. Comparison of the degradation rates of dG phosphoramidites with different protecting groups as well as with phosphoramidites containing bases that are structurally similar to dG affords clues to the nature of how dG catalyzes its own destruction and indicates a direct correlation between ease of protecting group removal and propensity to undergo autocatalytic degradation.
... L' intérêt de l' utilisation de la fonction phosphoramidite réside dans l' obtention d' un haut rendement de couplage avec une fonction hydroxyle, cette réaction étant bien connue dans la synthèse automatisée des oligonucléotides 33,34 (rendement > 99 % à chaque cycle de couplage). Ainsi, nous pourrons coupler le synthon sur une surface silanisée OH en vue de l' obtention d' une grande densité de fonctions oxyamines protégés par un groupe photosensible sur la surface du support solide ( Figure 16). ...
The aim of our work is to develop an effective method for the patterning of closed solid support such as microchannels or capillaries by biomolecules. The covalent attachment of biomolecules on the glass and silicon surfaces is carried out by using the formation an oxime linkage. Patterning is achieved upon illumination by deprotecting the aminooxy function protected by a photolabile (NPPOC) group. We have show that on both planar format and capillary format the aminooxy surface protected by a photolabile group, is effective for the immobilization of several oligonucleotides at predefined positions. The strategy was further extended for the immobilization of other molecules such as sugars, peptides or different hydrophobic and hydrophilic molecules in the capillaries. The results show the effectiveness of our strategy for the immobilization of variety of molecules. Further, we showed that the use of a hydrophilic surface (PEG) improves the sensitivity of this methodology.
... Dans les années 1980, Beaucage et Caruthers utilisèrent des monomères plus stables: les phosphoramidites (dérivés du phosphore trivalent à liaison P-N)(Beaucage & Iyer, 1992;Caruthers et al., 1980). En contre partie de cette stabilité, les phosphoramidites nécessitent une activation par un acide faible (tétrazole) pour réagir convenablement lors du couplage. ...
CLASSICAL ANALYSIS METHODS FOR ENZYMATIC DNA REPAIR ACTIVITY MONITORING ARE TEDIOUS AND TIME-CONSUMING (GEL ELECTROPHORESIS COUPLED TO ISOTOPE LABELING OR HIGH PERFORMANCE LIQUID CHROMATOGRAPHY). HENCE, WE DEVELOPED A NEW DNA REPAIR ASSAY BASED ON FRET (FLUORESCENT RESONANCE ENERGY TRANSFER) DETECTION. THIS HAS REQUIRED THE DESIGN OF AN ORIGINAL DNA PROBE: A HAIRPIN STRUCTURE CONTAINING SPECIFIC LESIONS LOCATED AT DEFINED SITES WITHIN THE STEM SEQUENCE, CLOSE TO THE CHROMOPHORE-LABELED EXTREMITIES. THE LESION EXCISION BY DNA N-GLYCOSYLASES LEADS TO THE COMPLEMENTARY STRAND SEPARATION, CAUSING A DROP IN FLUORESCENCE QUENCHING. THUS, THE CLEAVAGE EXTENT IS REFLECTED BY THE FLUOROPHORE EMISSION INTENSITY INCREASE AND STABILIZATION OVER THE REPAIR REACTION TIME-COURSE. AFTER PROVING THE LINEARITY OF THE ASSAY RESPONSE, WE DEMONSTRATED THE ABILITY OF THE CURRENT METHOD TO GIVE ACCESS TO REPAIR ENZYME KINETIC PARAMETERS. THE RELEVANCE OF THESE PARAMETERS WAS CHECKED BY COMPARISON TO VALUES, OBTAINED BY PAGE ANALYSES. THEN, WE INVESTIGATED THE CAPABILITY OF OUR TEST TO BE USED AS A HIGH-THROUGHPUT SCREENING TOOL TO QUANTIFY ENZYMATIC REPAIR ACTIVITIES AND CHECK INHIBITION EFFECTS (BY USING PURIFIED ENZYMES OR CELL EXTRACTS). FINALLY, A MINIATURIZATION PROJECT WAS CARRIED OUT TO IMPROVE THE SENSITIVITY OF THIS NEW DETECTION METHOD. THE PERFORMANCE OF THIS HOMOGENEOUS LIQUID-PHASE ASSAY SHOULD LEAD TO APPLICATIONS IN THE BIOANALYTICAL FIELD RELATED TO FUNDAMENTAL, BIOMEDICAL AND PHARMACEUTICAL RESEARCH.
... It is obvious that SM-FRET will require RNA molecules that are site-specifically labelled with donor and acceptor fluorophores for studying RNA conformational dynamics. Although phosphoramidite chemistry allows the site-specific introduction of a range of different modifications into short RNA, the same task remains a major challenge for larger RNAs (30)(31)(32)(33)(34). Furthermore, having multiple fluorophores even in short RNAs can be troublesome during solid-phase synthesis. ...
The oversimplified notion of RNA being a mere carrier of sequence information from gene to protein has been repeatedly undermined over the decades by yet another newly discovered function performed by certain RNA species. These new species include in particular RNAs which regulate gene expression in response to a metabolite sensing event. These RNAs — known as riboswitches — elegantly couple metabolite recognition with gene regulation in the apparent absence of protein helpers. Here we first sought to investigate the folding dynamics of the S-adenosyl-L-methionine responsive riboswitch by FRET spectroscopy. This requires the synthesis of full-length riboswitch constructs site-specifically modified with multiple fluorophores. For this challenging task we have established a 5-way splinted-ligation strategy to prepare dual-fluorophore labelled full-length riboswitch constructs in an unprecedented overall yield of 10 %. These constructs have further been subjected to bulk and single molecule FRET spectroscopy for ligand induced folding analysis. We confirmed similar folding dynamics for the aptamer of the complete riboswitch (aptamer + expression platform) as reported earlier for constructs containing the aptamer alone. However, we also observed a few other folding phenomena induced by a chemically slightly different, yet non-cognate metabolite, which cannot be explained by any facts known about this riboswitch to date and require further experiments to reach a final conclusion. During the course of the aforesaid work, we realized the limitations of existing nucleic acid functionalization strategies. Therefore we decided to use bioorthogonal click reactions as part of our labelling strategy. Among various different click reactions, we first had to find the one which best suits our purpose and to optimize its conditions. Having the optimized click reaction conditions at hand, we developed enzymatic strategies to site-specifically functionalize long RNAs with clickable residues. In our nucleic acid labelling strategy a diverse array of different chemical functionalities can be introduced exploiting the modular nature of click chemistry. This does not demand either de novo synthesis or optimizations of enzymatic reaction conditions for each new single compound. Furthermore, we developed a chemical approach using two different mutually orthogonal click reactions for concurrent, site-specific labelling of DNA molecules with multiple fluorophores. Moreover, we sought to extend this strategy of enzymatic, site-specific transfer of clickable residues to long RNAs towards photochemical transfer of clickable moieties to a target RNA in a mixture of many unrelated sequences. This technique, which we call Affinity-based Chemical RNomics is a chemical approach in experimental RNomics whereby RNA sequences which bind to a given small-molecule metabolite are to be isolated from a total RNA isolate of any organism just by the virtue of its tight binding to its cognate metabolite and without any prior knowledge of its sequence. This method would therefore allow for the discovery of previously unknown riboswitches, currently the only known kind of natural RNA that binds small-molecule metabolites. Since all currently known riboswitches have been discovered by rational approaches, this will considerably extend the chances of discovering new riboswitches.
Motivated by DNA storage systems, this work presents the DNA reconstruction problem, in which a length-n string, is passing through the DNA-storage channel, which introduces deletion, insertion and substitution errors. This channel generates multiple noisy copies of the transmitted string which are called traces. A DNA reconstruction algorithm is a mapping which receives t traces as an input and produces an estimation of the original string. The goal in the DNA reconstruction problem is to minimize the edit distance between the original string and the algorithm’s estimation. In this work, we present several new algorithms for this problem. Our algorithms look globally on the entire sequence of the traces and use dynamic programming algorithms, which are used for the shortest common supersequence and the longest common subsequence problems, in order to decode the original string. Our algorithms do not require any limitations on the input and the number of traces, and more than that, they perform well even for error probabilities as high as 0.27. The algorithms have been tested on simulated data, on data from previous DNA storage experiments, and on a new synthesized dataset, and are shown to outperform previous algorithms in reconstruction accuracy.
My thesis focused on the development of novel biomolecular NMR methodologies to characterise dynamics and interactions of biological macromolecules, by exploring novel horizons of biology, chemistry and NMR spectroscopy. My work first focused on micro-RNAs (miRNAs), key genetic regulators, capable to block messenger RNA (mRNA) translation. The let-7 miRNA and its targets were chemically synthesized to incorporate 19F probes, allowing to describe the interaction between let-7 and its target mRNAs by NMR. The second project was centered on a transfer RNA (tRNA), an essential component of the translation. Modulation of tRNA dynamic was probed using NMR 15N spin relaxation, in the context of tRNA maturation. Here, multiple-field relaxation rates, were measured and analysed to capture the fast dynamics of this system. The third study aimed at characterising a protein involved in the T5 bacteriophage infection of bacteria, and probe its interaction with its bacteria receptor protein. Resonance assignment of LLP the T5 protein involved in phage infection has been performed, as well as the 3D structure characterisation. Spin relaxation was used to probe LLP dynamics and titration experiments with the bacteria receptor allowed to reveal the binding interface. An additional project (not detailed) involved the dynamic study of an aquaporin, a membrane protein acting as water channel, by solid-state NMR at multiple fields. All these examples exemplify how NMR spectroscopy can probe complex dynamic processes in various contexts.
Mycotoxins are extremely dangerous, and their detection in our environment, food and feed is becoming increasingly important. Biosensors are being implemented heavily in mycotoxin detection along with other significant applications. Aptamers have numerous beneficial advantages as biorecognition molecules and are being used as the biorecognition part of biosensors (Aptasensors). The development of aptamers does not require inducing immune response against the target, but the SELEX method is used. The SELEX method is laborious, time consuming and can be expensive at times. Various efforts were done to replace that method with a computational alternative to reduce the effort, time and money needed to develop and design aptamers. One of the most significant efforts to achieve that was the MAWS algorithm. We used the MAWS algorithm to develop a new aptamer against aflatoxin B1, the most dangerous mycotoxin. The MAWS algorithm failed to function properly, and molecular modelling and molecular docking was used alternatively to achieve the same goal. A new pipeline for predicting ssDNA aptamers was proposed, a new aptamer against aflatoxin B1 was obtained and recommendations for further future research directions were given.
p>Inherited diversity in the human genome leads to phenotypic variations that underlie an individual’s response to their environment. By typing these differences, referred to as single nucleotide polymorphisms, on a whole genome basis, it is hoped that the complex genetic basis of susceptibility to common diseases can be elucidated.
Herein is described the basis of a potential assay for the determination of single nucleotide polymorphisms on a whole genome basis. Initial studies were focused on the ability of a resin bead to support oligonucleotide and peptide synthesis, oligonucleotide duplex formation and identification of a peptide by mass spectrometry from a minimum number of beads. Poor hybridisation kinetics led to a synthetic investigation into the ability to modify the properties of the oligonucleotides to improve the kinetics and thermodynamics of duplex association. Hereinafter, it is shown that the propargylamino or 3-aminoprop-1-yne group stabilises DNA duplexes when placed at the C-5 position in pyrimidines and C-7 position in 7-deazapurines.
At physiological pH, the cationic propargylamine group, when introduced into an oliognucleotide, helps to reduce the repulsion between the anions on the phosphodiester backbone and therefore reduces the salt dependence of duplex formation. The alkyne moiety increases the geometrical overlap and therefore the dispersion forces in single and double stranded DNA. The novel heterocyclic base 7-amino-1-ynyl-7-deaza-2,6-diaminopurine, when base paired with dT, has a similar thermodynamic stability to a G.C base pair. This novel adenine analogue may therefore by useful in applications where T <sub>m </sub>harmonisation would be an advantage.</p
Drug discovery from plants usually focuses on small molecules rather than such biological macromolecules as RNAs. Although plant transfer RNA (tRNA)-derived fragment (tRF) has been associated with the developmental and defense mechanisms in plants, its regulatory role in mammals remains unclear. By employing a novel reverse small interfering RNA (siRNA) screening strategy, we show that a tRF mimic (antisense derived from the 5 0 end of tRNA His(GUG) of Chinese yew) exhibits comparable anti-cancer activity with that of taxol on ovarian cancer A2780 cells, with a 16-fold lower dosage than that of taxol. A dual-luciferase reporter assay revealed that tRF-T11 directly targets the 3 0 UTR of oncogene TRPA1 mRNA. Furthermore, an Argonaute-RNA immunoprecipitation (AGO-RIP) assay demonstrated that tRF-T11 can interact with AGO2 to suppress TRPA1 via an RNAi pathway. This study uncovers a new role of plant-derived tRFs in regulating endogenous genes. This holds great promise for exploiting novel RNA drugs derived from nature and sheds light on the discovery of unknown molecular targets of therapeutics.
Understanding the function of oligonucleotides on a molecular level requires methods for studying their structure, conformational changes, and internal dynamics. Various biophysical methods exist to achieve this, including the whole toolbox of Electron Paramagnetic Resonance (EPR or ESR) spectroscopy. An EPR method widely used in this regard is Pulsed Electron-Electron Double Resonance (PELDOR or DEER), which provides distances in the nanometer range between electron spins in biomolecules with Angstrom precision, without restriction to the size of the biomolecule, and in solution. Since oligonucleotides inherently do not contain unpaired electrons, these have to be introduced in the form of so-called spin labels. Firstly, this protocol describes how nitroxide spin labels can be site-specifically attached to oligonucleotides using "Click" chemistry. The reaction provides little byproducts, high yields, and is conveniently performed in aqueous solution. Secondly, the protocol details how to run the PELDOR experiment, analyze the data, and derive a coarse-grained structure. Here, emphasis is placed on the pitfalls, requirements for a good dataset, and limits of interpretation; thus, the protocol gives the user a guideline for the whole experiment i.e., from spin labeling, via the PELDOR measurement and data analysis, to the final coarse-grained structure. Graphical abstract: Schematic overview of the workflow described in this protocol: First, the spin-labeling of RNA is described, which is performed as a "Click"-reaction between the alkyne-functionalized RNA strand and the azide group of the spin label. Next, step-by-step instructions are given for setting up PELDOR/DEER distance measurements on the labeled RNA, and for data analysis. Finally, guidelines are provided for building a structural model from the previously analyzed data.
A risk-based approach for routine identity testing of therapeutic oligonucleotide drug substances and drug products is described. Risk analysis of solid-phase oligonucleotide synthesis indicates that intact mass measurement is a powerful technique for confirming synthesis of the intended oligonucleotide. Further risk assessment suggests that the addition of a second, sequence-sensitive identity test, which relies on a comparison of some property of the sample to a reference standard of proven identity, results in a sufficient test of identity for most oligonucleotide drug substances and products. Alternative strategies for drug product identity testing are presented. The analysis creates a common way to communicate risk and should result in a harmonized approach to identity testing that avoids the unnecessary analytical burden associated with routine de novo sequencing, without compromising quality or patient safety.
The efficiency of Polymerase Chain Reaction (PCR) depends on multiple factors. The primers are very important components of PCR which determine the specificity of amplification. In this review, considerable attention is paid to the principles of selection of primers and the requirements for their design with the help of appropriate software. The problems of elimination of primer dimers, as well as the thermodynamics of DNA duplex structures and their melting temperatures, largely dependent not only on the GC-composition, but also on the stacking interactions of neighboring nucleotides, are considered. The applicability of the NN-model of DNA thermodynamics to determine the melting and annealing temperatures of primers is shown. Various formulas for calculating these temperatures are given. The so-called degenerate primers, primers with extra-sequences at the 5'-end, modified and discriminating primers are described separately. The topics of chemical and enzymatic synthesis of oligonucleotides, which also include historical aspects, are also discussed.
This review selects some representative coupling agents used for internucleotide bond formation reactions in the phosphoramidite method, which is now the most widely employed method for the chemical synthesis of oligodeoxyribonucleotides and oligoribonucleotides, and it describes their utility, efficiency, and drawbacks. Moreover, the mechanism of the coupling of the nucleoside phosphoramidite and nucleoside promoted by the coupling agent is discussed in some cases. The selected coupling agents are 1H-tetrazole, 5-ethylthio-1H-tetrazole (ETT), 5-benzylthio-1H-tetrazole (BTT), 5-[3,5-bis(trifluoromethyl)phenyl]-1H-tetrazole (Activator 42), 4,5-dicyanoimidazole (DCI), certain carboxylic acids, and various acid/azole complexes such as benzimidazolium triflate (BIT) and saccharin 1-methylimidazole (SMI).
Recent studies on the chemical synthesis of RNA and related derivatives are reviewed. In particular, a variety of new 2′-hydroxyl protecting groups that are developed during the past decade are described and compared with the conventional ones from the organochemical point of view. Great improvements in the coupling efficiency and suppression of side reactions during RNA synthesis cycles are described in great detail. The methods and associated problems for constructing the key synthetic intermediates, i.e., 2′-O-protected ribonucleoside 3′-phosphoramidite building blocks, are also discussed.
Introductory paragraph
DNA-based data storage has emerged as a promising method to satisfy the exponentially increasing demand for information storage. However, practical implementation of DNA-based data storage remains a challenge because of the high cost of DNA per unit data. Here, we propose the use of eleven degenerate bases as encoding characters in addition to A, C, G, and T, which increases the information capacity (the amount of data that can be stored per length of DNA sequence designed) and reduce the cost of DNA per unit data. Using the proposed method, we experimentally achieved an information capacity of 3.37 bits/character, which is more than twice when compared to the highest information capacity previously achieved. Finally, the platform was projected to reduce the cost of DNA-based data storage by 50%.
Oligonucleotides serve as important tools for biological, chemical, and medical research. The preparation of oligonucleotides through automated solid-phase synthesis is well-established. However, identification of byproducts generated from DNA synthesis, especially from oligonucleotides containing site-specific modifications, is sometimes challenging. Typical high-performance liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoresis methods alone are not sufficient for characterizing unexpected byproducts, especially for those having identical or very similar molecular weight (MW) to the products. We used a rigorous quality control procedure to characterize byproducts generated during oligonucleotide syntheses: (1) purify oligonucleotides by different HPLC systems; (2) determine exact MW by high-resolution MS; (3) locate modification position by MS/MS or exonuclease digestion with matrix-assisted laser desorption ionization-time of flight analysis; and (4) conduct, where applicable, enzymatic assays. We applied these steps to characterize byproducts in the syntheses of oligonucleotides containing biologically important methyl DNA adducts 1-methyladenine (m1A) and 3-methylcytosine (m3C). In m1A synthesis, we differentiated a regioisomeric byproduct 6-methyladenine, which possesses a MW identical to uncharged m1A. As for m3C, we identified a deamination byproduct 3-methyluracil, which is only 1 Da greater than uncharged m3C in the ∼4900 Da context. The detection of these byproducts would be very challenging if the abovementioned procedure was not adopted.
We describe a total synthesis of a polyunsaturated fatty acid (PUFA)-containing glucuronosyldiacylglycerol (GlcADG), which is a surrogate glycolipid whose synthesis is remarkably upregulated in plant membranes under phosphorus-depleted conditions. Glycosylation between the glucuronide donor bearing 3,4-dimethoxybenzyl (DMPM) protecting groups and di-acylglycerol acceptor proceeded smoothly in the presence of gold(I) catalyst to provide the protected α-isomer of GlcADG as the major product.
The acetyl capping reaction used throughout solid phase oligonucleotide synthesis is meant to minimize n−1 deletionmer impurities by terminating sequences that fail to couple to a phosphoramidite. However, the reaction is also responsible for the formation of a number of impurities. One capping-related impurity has an additional mass of 98 amu from the parent oligonucleotide. The n+98 amu impurity was found to result from modification of an adenine nucleobase. The structure of the impurity was determined by preparation of an oligonucleotide enriched in n+98 amu, enzymatic digestion to individual nucleosides, isolation of the pure nucleoside+98 amu species, crystallization, and X-ray crystallographic analysis. The n+98 amu impurity is an oligonucleotide in which one adenine residue has been converted to 5-amino-4-pyrimidinylimidazole. The mechanism of formation of the impurity was investigated, and a mechanism is proposed.
Due to their high potency (EC50 ~1 pM) and exquisite target selectivity for all expressed mRNAs, small interfering RNA (siRNA)-induced RNAi responses hold significant promise as a therapeutic modality. However, the size and high negative charge of siRNAs render them unable to enter cells without assistance from a delivery agent. Most current methods of siRNA delivery rely on encasing siRNA molecules in large nanoparticles or cationic liposomes. However, these approaches suffer from a number of problems, including a poor diffusion coefficient, cytotoxicity, and poor pharmacokinetics. To address the problem of siRNA in vivo delivery, we developed monomeric neutral RNAi prodrugs, termed siRibonucleic neutrals (siRNNs), that directly neutralize the phosphate backbone negative charge by synthesis with bioreversible phosphotriester groups that are enzymatically cleaved offin the cytoplasm of cells. Here we describe the synthesis and purification of siRNN conjugates that induce in vivo target gene knockdown following systemic delivery into mice.
"Smart" materials are polymer systems that are able to change their physical or chemical properties in response to external stimuli in their environment. By adding a specific molecular recognition probe to a polymer, hybrid materials can be developed that retain the properties of the advanced polymer and gain the ability to respond to a specific molecular target. Aptamers are single-stranded oligonucleotides that are well-suited to serve as molecular recognition probes due to the specificity and affinity of their target recognition as well as their stability and ease of synthesis and labeling. In particular, their negatively charged backbone makes for their facile incorporation into polyelectrolyte-based materials. This article will provide a brief review of the currently reported biosensor and delivery platforms that have been reported employing aptamer-polyelectrolyte materials, as well as a detailed description of the methods used to synthesize and study films and microcapsules containing small-molecule aptamer probes.
The fifth edition continues in the tradition of the previous volumes. The literature search is complete to the middle of 2013, and was done using a hand search where I looked at the individual papers to find appropriate material and by using the search engines provided by the various publishers. SciFinder was also used to complement my search, by looking for specific information rather than a general search of protective group chemistry as this results in too many hits to examine. Given the ever-expanding literature, it is becoming increasingly more time consuming to maintain the comprehensive tradition of the last four editions. If I have passed over a favorite method or even a new protective group, it was not done intentionally. During the preparation of this edition, I processed over 4100 new references. Not all have been included because in many cases the examples did not offer anything new. However, approximately 2800 new references have been included in this edition. Overall, I have tried to be as all-inclusive as possible because this book is about giving the user all the available options for protection and deprotection. Protective group chemistry is largely driven by natural product synthesis, and over the years since the last edition, the emphasis on highly hydroxylated natural products has given way to more alkaloid natural products that tend not to use protective groups as heavily. In fact, there are many syntheses that have avoided the use of protective groups altogether. There are, however, many classes of molecules where our chemical technology is still not adequate to completely avoid the use of protective groups, such as in polypropionate macrolide synthesis, peptide synthesis, and oligonucleotide synthesis.
DNA which is the support of genetic information is constantly subjected to damaging agents. These processes can lead to structural modifications of the DNA molecule and harmful biological consequences, such as mutagenesis or cancer. DNA damages can be repaired by enzymatic complexes which restore the original sequence of the biopolymer. In this study we were interested in structural aspects of DNA lesions and in their repair by the base excision repair (BER) and the reversion repair (RR) pathways. The present work consisted in developing a new tool in a biochip format aim at detecting these repairs activities by fluorescence measurement. Thus, hairpin-shaped damaged oligonucleotides were grafted on glass slides by a click chemistry approach. After optimization of the immobilization conditions, by click chemistry, this new biosensor was validated for the detection of repair activities using purified enzymes (glycosylases and AP-endonucleases) and cell extracts. Using a similar approach, we adapted this microarray to measure reversion repair and to screen DNA repair inhibitors. In a second part of this work, we applied the pulsed electron paramagnetic resonance (pulsed EPR) technique to study structural deformation induced by several DNA damages. To achieve this goal we developed a multiple site-specific labelling procedure of the DNA probes by nitroxide radicals. Then, pulsed EPR was used for the first time to detect an enzymatic DNA repair activity.
Detection of nucleic acids in whole tissues has become key in our understanding of gene expression during development. In situ hybridization (ISH) has been an invaluable technique in the making of numerous discoveries. Most recently, the technical advance of using short, fluorescently labeled probes has allowed for the detection of single-mRNA molecules. Thus, quantification of RNA levels in single cells or even within subcellular regions is now possible without RNA isolation. In combination with the immunofluorescence (IF) technique, visualization of nucleic acids and associating proteins is achieved with higher resolution than ever before using light microscopy. Here we describe the steps implemented to achieve the visualization of individual messenger RNAs (mRNA) using single-molecule FISH (smFISH) probes, as well as detection of mRNA/protein (mRNP) complexes via smFISH in combination with IF.
The monomer units of a polymer can be used to encode a message. This property is used, for instance, by nature to store genetic information in DNA macromolecules. Therefore, during the past decades, many researchers have aimed to recreate in vitro or in vivo the properties of nucleic acids. Peptide nucleic acids, or more generally speaking xeno-nucleic acids, are interesting examples of man-made genetic polymers. However, the genetic code is surely not the only type of code that can be “written” in a polymer. In principle, many other monomer-based codes could be developed. For example, a binary code can be potentially implemented in a synthetic macromolecule using two comonomers defined as 0 and 1 bit. This possibility is exciting because it would permit to develop a full new class of synthetic polymers, which contain sequence-coded information. Such polymers could be interesting for a variety of new applications, for example in the field of data storage and product identification. However, these tempting options are currently underexplored. It should be clarified that the development of information-containing macromolecules is not trivial. First of all, in order to contain “readable” information, such polymers should possess perfectly controlled comonomer sequences. Moreover, chemical and analytical methods that allow deciphering of sequence-coded information have to be developed. The aim of the present Perspective is to show that significant progress has been done in that direction during the past two years. For instance, convenient strategies have been reported for the preparation of monodisperse sequence-defined macromolecules. In addition, encouraging advances have been made for the sequencing of non-natural polymers. These recent results are discussed and critically analyzed herein. Altogether, monomer-based information storage should be regarded as a new property of synthetic matter.
The synthesis of pure TCB-dichlorophosphite and di-TCB-monochlorophosphite are described, as well as the formation of the di-triazolide from TCB-dichlorophosphite and its use in the preparation of a dinucleotide derivative.
In the presence of 1-methylimidazole, 2--acyl guanine (as in ), thymine (as in ) and uracil (as in ) residues react readily with the phosphorylating agent derived from 2-chlorophenyl phosphorodichloridate () and 1-hydroxybenzotriazole.
Several advances in the chemistry of solid-phase oligonucleotide synthesis are described. In the deoxy series the 4-phenylazophenyloxycarbonyl group can be selectively introduced at the 5 prime -position and cleaved by transesterification/ beta -elimination. With this substituent acid deprotection, i. e. risk of depurination, can be avoided during chain elongation. As new tools for oligonucleotide synthesis immobilized nucleoside phosphoramidites have been prepared as polymeric reagents. The activated nucleotides released from these reagents by tetrazole/acetonitrile were shown to give excellent yields in the elongation of support-bound oligonucleotides ('three-phase synthesis'). Implications of this reaction on the mechanism of phosphite triester synthesis are discussed.
A new approach to the synthesis of 2′(3′)-O-aminoacyl oligoribonucleotides which are the 3′-terminal sequences of corresponding aa-tRNAs is described. It is exemplified by the synthesis of the trinucleotide sequence C-C-A-Phe (10). This compound was synthesized by employing the benzotriazolyl phosphotriester approach using the following protecting groups on the components: a (9-fluorenylmethyloxy)carbonyl for the cytosine and adenine amino groups; 2-chlorophenyl groups for the internucleotide bond protection; a dimethoxytrityl group for blocking of the 5′-hydroxy function; a 4-methoxytetrahydropyranyl group for protection of 2′-hydroxy functions; and a (2-(4-biphenylyl)isopropyloxy)carbonyl group for blocking of the α-amino acid. Protected C-C-A (7) was synthesized in a stepwise fashion and quantitatively aminoacylated with ((2-(4-biphenylyl)isopropyloxy)carbonyl)-L-phenylalanine in the presence of mesitylenesulfonyltetrazole. The resulting fully blocked derivative 9 was deprotected in only two steps via oximate and diluted formic acid treatment to obtain the desired C-C-A-Phe (10) in ca. 30% isolated yield by using a new isolation procedure. Thus this approach has potential to become a general method for the synthesis of various 2′(3′)-O-aminoacyl oligoribonucleotides which are important tools for mechanistic studies of protein synthesis in in vitro systems.
The phosphotriester approach is employed for the synthesis. The complementarity of two acid-labile groups, 9-phenylxanteh-9-yl- and 4-methoxytetrahdropyranyl-, which have been used for the protections of 5' and 2'-hydroxyl functions, respectively, has been demonstrated through the present work. The fluoren-9-methyl-(FM) group has also been introduced, through the present work, to oligoriobonucleotide chemistry for the protection of a 3'-phosphodiester to the level of a neutral 3'-phosphotriester, which made it possible to carry out a block synthesis strategy for the undecaribonucleotide sequence.
Uridine derivatives are protected at N3-position with a p-methoxybenzyl group under Mitsunobu conditions. Selective removal is possible with ceric ammonium nitrate. The otherwise difficult to obtain 5'-O-phosphonomethyl d4U was prepared using this strategy.
Umsetzung des Disiloxans (I) mit den Titelverbindungen (II) bzw. (IV) liefert die Derivate (III) bzw. (V), die u.a. anhand von O-Acylderivaten charakterisiert werden.
Four ribonucleoside phosphoramidites bearing 5'-O-dimethoxytrityl and 2'-O-tetrahydrofuranyl groups were synthesized with the use of bis(diisopropylamino)-methoxyphosphine in yields of 68-92%. By the same procedure, the phosphoramidite derivatives of dimers were also prepared for block condensation.We measured the time course of the condensation reaction of ribonucleoside phosphoramidite and nucleoside linked to long-chain alkylamine-controlled pore glass (LCA-CPG) resin. It took 30 min for complete coupling if the nucleoside phosphoramidite was activated with 1-H-tetrazole. However, when 5-p-nitrophenyltetrazole was used for the activation, the condensation reaction was complete within 5 min. The behavior of the block condensation reaction using dimers was similar to that of monomers in the case of 5-p-nitrophenyltetrazole activation.We synthasized oligoribonucleotides UUUU, CUCUCUU and GGCGCGCC in yields of 51, 33 and 3%, respectively, by the phosphite-triester approach by means of either stepwise or block condensation on the LCA-CPG resin.
2'-0-Tetrahydrofuranyl nucleosides have been synthesized from 2,3-dihydrofuran via 3',5'-bis-tert-butyldimethylsilylnucleosides. These nucleosides were used as intermediates for oligonucleotide syntheses by the phosphotriester method. Trimers C-C-A and C-C-U were synthesized by the stepwise addition of monomers. U-U-U-U-U-U, A-U-G-A-U-G and A-U-G-A-U-G-A-U-G were obtained by the block condensation of trimers in which the 5'-hydroxy-l group had been selectively phosphorylated. © 1983, The Pharmaceutical Society of Japan. All rights reserved.
A new type of silyl protecting group was designed and a reagent for its introduction was obtained. This reagent, 1,3--dihalo-1,1,3,3-tetraalkyldisiloxane,
allows simultaneous protection of 3′-OH and 5′-OH groups of nucleoside in high yield. The properties of the new protecting
group were studied and some aspects of its applications are presented.
Ribooligonucleotides (nonamer, decamer and nonadecamer) were synthesized by the solid phase phosphotriester method using 2'-O-tetrahydrofuranyl nucleosides. These oligomers were designed in order to obtain E. coli tRNAMetf deficient in D-loop and D-stem and its analogs. Condensation was performed by using dimer blocks and the dimethoxytrityl group on the 5'-hydroxyl group was removed selectively by 1M ZnBr2 without affecting the 2'-protection.
The protection of the O6-amide group of deoxyguanosine with the 3, 4-methoxybenzyl group is described. This protecting group could be introduced effectively and was readily removed with DDQ. This was used to demonstrate the synthesis of the oligodeoxyribonucleotide.
A nonaribonucleotide CAGGUAAGU has been synthesized by the phosphotriester solid-phase method. The 5'-linked 2'-O-tetrahydrofuranyl-N-benzoylcytidine 3'-O-(o-chlorophenyl) phosphate was used as the starting material and the chain was elongated in the 3'-direction by removal of the phosphoro-p-anisidate group using isoamyl nitrite under neutral conditions.
Using CH3PCl2 and suitably protected nucleosides at low temperature remarkable diastereoselectivity is observed in the synthesis of dinucleoside methylphosphonates.
N-Acyl protecting group in nucleoside derivatives can be selectively removed by treatment with zinc bromide in the presence of alcohols to give O-protected nucleosides.
Barium salts of protected mono- and dinucleotides have been employed in a scheme for the construction of oligodeoxyribonucleotides on a glass support. The excesses of these synthetic units were recovered simply by precipitation, and reused in subsequent cycles.
DMTrTp(SPh)2 was proposed as a simplest “nucleotide unit” for the synthesis of olygothymidylates. One phenylthio group was removed selectively and rapidly from dithiol-esters by 1 M pyridinium hypophosphonate. Two kinds of arenedisulfonyl chlorides were newly prepared and utilized as promising condensing agents.
The suitability of the 4-methoxytetrahydropyran-4-yl group for the protection of 2′ (or 3′)-hydroxy functions in oligoribonucleotide synthesis is confirmed; the latter protecting group is removed in 0.01M - hydrochloric acid at room temperature under conditions which, contrary to a recent report, lead to no detectable cleavage or migration of the internucleotide phosphodiester linkages.
The stepwise synthesis of a nonadecaribonucleotide corresponding to units 9 to 27 of the t·RNAfmet from is described. The chlorophoshite condensation procedure and a silica gel support were used.
The oligoribonucleotides (Ap)7A, (CP)7C, (Gp)7G and (UP)7U were prepared via an automated synthesizer using a silica gel support and the methyl dichlorophosphite procedure.
-nonacylated nucleoside phosphoramidites can be prepared in two steps from 2′-deoxynucleosides. Their use in phosphorylation reactions is exemplified.
The large scale synthesis of two octadeoxyribonuclcotides has been successfully accomplished on polystyrene by the phosphite triester approach. Employing moderate excesses but high concentrations of phosphoramidites, the coupling yields are similar to those obtained in standard small scale syntheses.
A new route, using phosphotriester intermediates, for the synthesis of branched ribonucleotides is described.