Steven Benner

• Manager at University of Florida

Anthropogenic evolvable genetic information systems (AEGIS) are DNA-like molecules that can be copied, support laboratory in vitro evolution (LIVE), and evolve to give AegisBodies, analogs of antibodies. However, unlike DNA aptamers built from four different nucleotides, AegisBodies are currently built from six different nucleotides. Thus, six-lett...

Metagenomic sequencing and data analysis of Arctic Ocean marine sediments to determine the microbial composition of these extreme environments. Once the data set is completed this will lay the foundation for nutrient cycling and potential climate change effects on the microbial biodiversity. Further, these organisms can be used as analogs for study...

Artificially expanded genetic information systems (AEGIS) were developed to expand the diversity and functionality of biological systems. Recent experiments have shown that these expanded DNA molecular systems are robust platforms for information storage and retrieval as well as useful for basic biotechnologies. In tandem, nucleic acid nanotechnolo...

A route to prepare ribonucleoside triphosphates featuring a 3’-aminoxy (3’-O-NH 2 ) removable blocking group is reported here. We then show that versions of two DNA polymerases, human DNA polymerase theta (Polθ) and mimiviral PrimPol, accept these triphosphates as substrates to add single nucleotides to an RNA primer under engineered conditions. Cl...

Environmental surveillance and clinical diagnostics heavily rely on the polymerase chain reaction (PCR) for target detection. A growing list of microbial threats warrants new PCR-based detection methods that are highly sensitive, specific, and multiplexable. Here, we introduce a PCR-based icosaplex (20-plex) assay for detecting 18 enteropathogen an...

We present the draft genome of the bacterium Thermoanaerobacter thermohydrosulfuricus strain AK152, a thermophilic, endospore–spore-forming, anaerobe isolated from a hot spring in Grensdalur, in Southwestern Iceland. This assembled genome will lay the foundation for identifying the carboxylic and amino acid fermentation pathways, suggesting biotech...

Nanopores are increasingly powerful tools for single molecule sensing, in particular, for sequencing DNA, RNA and peptides. This success has spurred efforts to sequence non-canonical nucleic acid bases and amino acids. While canonical DNA and RNA bases have pKas far from neutral, certain non-canonical bases, natural RNA modifications, and amino aci...

Adding synthetic nucleotides to DNA increases the linear information density of DNA molecules. Here we report that it also can increase the diversity of their three-dimensional folds. Specifically, an additional nucleotide (dZ, with a 5-nitro-6-aminopyridone nucleobase), placed at twelve sites in a 23-nucleotides-long DNA strand, creates a fairly s...

Arthropod-borne viruses are major causes of human and animal disease, especially in endemic low-and middle-income countries. Mosquito-borne pathogen surveillance is essential for risk assessment and vector control responses. Sentinel chicken serosurveillance (anti-body testing) and mosquito pool screening (by RT-qPCR or virus isolation) are current...

With just four building blocks, low sequence information density, few functional groups, poor control over folding, and difficulties in forming compact folds, natural DNA and RNA have been disappointing platforms from which to evolve receptors, ligands, and catalysts. Accordingly, synthetic biology has created “artificially expanded genetic informa...

Ribose is the defining sugar in ribonucleic acid (RNA), which is often proposed to have carried the genetic information and catalyzed the biological reactions of the first life on Earth. Thus, abiological processes that yield ribose under prebiotic conditions have been studied for decades. However, aqueous environments required for the formation of...

Pathological hyperphosphorylation and aggregation of microtubule‐associated Tau protein contribute to Alzheimer's Disease (AD) and other related tauopathies. Currently, no cure exists for Alzheimer's Disease. Aptamers offer significant potential as next‐generation therapeutics in biotechnology and the treatment of neurological disorders. Traditiona...

Arthropod-borne viruses are major causes of human and animal disease, especially in endemic low- and middle-income countries. Mosquito-borne pathogen surveillance is essential for risk assessment and vector control responses. Sentinel chicken serosurveillance (antibody testing) and mosquito pool screening (by RT-qPCR or virus isolation) are current...

Pathological hyperphosphorylation and aggregation of microtubule‐associated Tau protein contribute to Alzheimer's Disease (AD) and other related tauopathies. Currently, no cure exists for Alzheimer's Disease. Aptamers offer significant potential as next‐generation therapeutics in biotechnology and the treatment of neurological disorders. Traditiona...

Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting “preferred orientations” on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution at...

Artificially Expanded Genetic Information Systems (AEGIS) add independently replicable unnatural nucleotide pairs to the natural G:C and A:T/U pairs found in native DNA, joining the unnatural pairs through alternative modes of hydrogen bonding. Whether and how AEGIS pairs are recognized and processed by multi-subunit cellular RNA polymerases (RNAPs...

The 4-letter DNA alphabet (A, T, G, C) as found in Nature is an elegant, yet non-exhaustive solution to the problem of storage, transfer, and evolution of biological information. Here, we report on strategies for both writing and reading DNA with expanded alphabets composed of up to 12 letters (A, T, G, C, B, S, P, Z, X, K, J, V). For writing, we d...

We show that in silico design of DNA secondary structures is improved by extending the base pairing alphabet beyond A-T and G-C to include the pair between 2-amino-8-(1'-β-d-2'-deoxyribofuranosyl)-imidazo-[1,2-a]-1,3,5-triazin-(8H)-4-one and 6-amino-3-(1'-β-d-2'-deoxyribofuranosyl)-5-nitro-(1H)-pyridin-2-one, abbreviated as P and Z. To obtain the t...

Many studies have suggested that the oxidized form of nicotinamide adenine dinucleotide (NAD⁺) is involved in an extensive spectrum of human pathologies, including neurodegenerative disorders, cardiomyopathy, obesity, and diabetes. Further, healthy aging and longevity appear to be closely related to NAD⁺ and its related metabolites, including nicot...

Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting “preferred orientations” on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution at...

Recently reported "displaceable probe" loop amplification (DP-LAMP) architecture has shown to amplify viral RNA from SARS-CoV-2 with little sample processing. The architecture allows signals indicating the presence of target nucleic acids to be spatially separated, and independent in sequence, from the complicated concatemer that LAMP processes cre...

NAD+ is known classically as a metabolite that participates in catabolic and anabolic pathways throughout the metabolism that is taught to students in introductory biochemistry courses. However, non-classical studies starting over a decade ago found that NAD+ is also involved in higher order functions, in part because of its involvement in the acti...

We show that in silico design of DNA secondary structures is improved by extending the base pairing alphabet beyond A-T and G-C to include the pair between 2-amino-8-(1'-β-D-2'-deoxyribofuranosyl)-imidazo-[1,2-a]-1,3,5-triazin-(8H)-4-one and 6-amino-3-(1'-β-D-2'-deoxyribofuranosyl)-5-nitro-(1H)-pyridin-2-one, simply P and Z. To obtain the thermodyn...

One horizon in synthetic biology seeks alternative forms of DNA that store, transcribe, and support the evolution of biological information. Here, hydrogen bond donor and acceptor groups are rearranged within a Watson-Crick geometry to get 12 nucleotides that form 6 independently replicating pairs. Such artificially expanded genetic information sys...

Chemists have now synthesized new kinds of DNA that add nucleotides to the four standard nucleotides (guanine, adenine, cytosine, and thymine) found in standard Terran DNA. Such "artificially expanded genetic information systems" are today used in molecular diagnostics; to support directed evolution to create medically useful receptors, ligands, an...

The first structural model of duplex DNA reported in 1953 by Watson & Crick presented the double helix in B-form, the form that genomic DNA exists in much of the time. Thus, artificial DNA seeking to mimic the properties of natural DNA should also be able to adopt B-form. Using a host–guest system in which Moloney murine leukemia virus reverse tran...

Reviewed are three decades of synthetic biology research in our laboratory that has generated alternatives to standard DNA and RNA as possible informational systems to support Darwinian evolution, and therefore life, and to understand their natural history, on Earth and throughout the cosmos. From this, we have learned that: • the core structure of...

The ability of nucleic acids to catalyze reactions (as well as store and transmit information) is important for both basic and applied science, the first in the context of molecular evolution and the origin of life and the second for biomedical applications. However, the catalytic power of standard nucleic acids (NAs) assembled from just four nucle...

The composition, sizes and shapes of particles in the clouds of Venus have previously been studied with a variety of in situ and remote sensor measurements. A number of major questions remain unresolved, however, motivating the development of an exploratory mission that will drop a small probe, instrumented with a single-particle autofluorescence n...

The composition, sizes and shapes of particles in the clouds of Venus have previously been studied with a variety of in situ and remote sensor measurements. A number of major questions remain unresolved, however, motivating the development of an exploratory mission that will drop a small probe, instrumented with a single-particle autofluorescence n...

A fundamental property of DNA built from four informational nucleotide units (GCAT) is its ability to adopt different helical forms within the context of the Watson-Crick pair. Well-characterized examples include A-, B-, and Z-DNA. For this study, we created an isoinformational biomimetic polymer, built (like standard DNA) from four informational "...

Boron (B) is considered a prebiotic chemical element with a role in both the origin and evolution of life, as well as an essential micronutrient for some bacteria, plants, fungi, and algae. B has beneficial effects on the biological functions of humans and animals, such as reproduction, growth, calcium metabolism, bone formation, energy metabolism,...

Before the first humans depart for Mars in the next decade, hundreds of tons of martian water-ice must be harvested to produce propellant for the return vehicle, a process known as in situ resource utilization (ISRU). We describe here an instrument, the Agnostic Life Finder (ALF), that is an inexpensive life-detection add-on to ISRU. ALF exploits a...

Boron (B) is considered a prebiotic chemical element with a role in both the origin and evolution of life, as well as an essential micronutrient for plants, some bacteria, fungi, and algae. B has beneficial effects on the biological functions of humans and animals, such as reproduction, growth, calcium metabolism, bone formation, energy metabolism,...

Reported here are experiments that show that ribonucleoside triphosphates are converted to polyribonucleic acid when incubated with rock glasses similar to those likely present 4.3-4.4 billion years ago on the Hadean Earth surface, where they were formed by impacts and volcanism. This polyribonucleic acid averages 100-300 nucleotides in length, wit...

Water-in-oil emulsions provide matrices for compartments that have many uses in diversity science. However, hydrophobic species are frequently incompatible with biological systems. For this reason, fluorinated matrices are often sought, since fluorinated species are neither hydrophilic nor hydrophobic; they therefore do not interact with most biomo...

Managing the pandemic caused by SARS-CoV-2 requires new capabilities in testing, including the possibility of identifying, in minutes, infected individuals as they enter spaces where they must congregate in a functioning society, including workspaces, schools, points of entry, and commercial business establishments. Here, the only useful tests (a)...

While nucleoside 5'-triphosphates are precursors for RNA in modern biology, the presumed difficulty of making these triphosphates on Hadean Earth has caused many prebiotic researchers to consider other activated species for the prebiotic synthesis of RNA. We report here that nickel(II), in the presence of borate, gives substantial amounts (2-3%) of...

Recently we showed the reduction and oxidation of six natural 2′-deoxynucleosides in the presence of the ambient oxygen using the very broad potential window of a pyrolytic graphite electrode (PGE). Using the same procedure, 2′-deoxynucleoside analogs (dNs) that are parts of an artificially expanded genetic information system (AEGIS) were analyzed....

DNA polymerases are today used throughout scientific research, biotechnology and medicine, in part for their ability to interact with unnatural forms of DNA created by synthetic biologists. Here especially, natural DNA polymerases often do not have the "performance specifications" needed for transformative technologies. This creates a need for scie...

Managing the pandemic caused by SARS-CoV-2 requires new capabilities in testing, including the possibility of identifying, in minutes, infected individuals as they enter spaces where they must congregate in a functioning society, including workspaces, schools, points of entry, and commercial business establishments. Here, the only useful tests (a)...

NASA should design missions to Mars for the purpose of generating "Aha!" discoveries to jolt scientists contemplating the molecular origins of life. These missions should be designed with an understanding of the privileged chemistry that likely created RNA prebiotically on Earth, and universal chemical principles that constrain the structure of Dar...

The Front Cover summarizes the discussions of a path‐hypothesis to make prebiotic RNA, via mineral‐guided maturation of carbonyl species and base precursor formation in a post‐impact reduced atmosphere, presented in the review.More information can be found in the Review by S. A. Benner et al.

“In a workshop in October 2018 organized by the Foundation for Applied Molecular Evolution and funded by the John Templeton Foundation, a group of planetary, biological, and molecular scientists, through open discussion of several paradoxes, resolved a path‐hypothesis to make prebiotic RNA, as summarized in the cover image and detailed in the revie...

Despite its widespread value to molecular biology, the polymerase chain reaction (PCR) encounters modes that unproductively consume PCR resources and prevent clean signals, especially when high sensitivity, high SNP discrimination, and high multiplexing are sought. Here, we show how "self-avoiding molecular recognition systems" (SAMRS) manage such...

The Varkud satellite ribozyme catalyses site-specific RNA cleavage and ligation, and serves as an important model system to understand RNA catalysis. Here, we combine stereospecific phosphorothioate substitution, precision nucleobase mutation and linear free-energy relationship measurements with molecular dynamics, molecular solvation theory and ab...

Expanding the number of nucleotides in DNA increases the information density of functional DNA molecules, creating nanoassemblies that cannot be invaded by natural DNA/RNA in complex biological systems. Here, we show how six‐letter GACTZP DNA contributes this property in two parts of a nanoassembly: 1) in an aptamer evolved from a six‐letter DNA li...

We present a direct route by which RNA might have emerged in the Hadean from a fayalite–magnetite mantle, volcanic SO2 gas, and well-accepted processes that must have created substantial amounts of HCHO and catalytic amounts of glycolaldehyde in the Hadean atmosphere. In chemistry that could not not have happened, these would have generated stable...

Expanding the number of nucleotides in DNA increases the information density of functional DNA molecules, creating nanoassemblies that cannot be invaded by natural DNA/RNA in complex biological systems. Here, we show how 6‐letter GACTZP DNA contributes this property in two parts of a nanoassembly: (1) in an aptamer evolved from a 6‐letter DNA libra...

The widespread presence of ribonucleic acid (RNA) catalysts and cofactors in the Earth′s biosphere today suggests that RNA was the first biopolymer to support Darwinian evolution. However, most “path‐hypotheses” to generate building blocks for RNA require reduced nitrogen‐containing compounds not made in useful amounts in the CO2−N2−H2O atmospheres...

The widespread presence of ribonucleic acid (RNA) catalysts and cofactors in Earth's biosphere today suggests that RNA was the first biopolymer to support Darwinian evolution. However, most "path-hypotheses" to generate building blocks for RNA require reduced nitrogen-containing compounds not made in useful amounts in the CO2-N2-H2O atmospheres of...

Background: The global expansion of dengue (DENV), chikungunya (CHIKV), and Zika viruses (ZIKV) is having a serious impact on public health. Because these arboviruses are transmitted by the same mosquito species and co-circulate in the same area, a sensitive diagnostic assay that detects them together, with discrimination, is needed. Methods: We...

Expanding the genetic code DNA and RNA are naturally composed of four nucleotide bases that form hydrogen bonds in order to pair. Hoshika et al. added an additional four synthetic nucleotides to produce an eight-letter genetic code and generate so-called hachimoji DNA. Coupled with an engineered T7 RNA polymerase, this expanded DNA alphabet could b...

Synthetic biologists demonstrate their command over natural biology by reproducing the behaviors of natural living systems on synthetic biomolecular platforms. For nucleic acids, this is being done stepwise, first by adding replicable nucleotides to DNA, and then removing its standard nucleotides. This challenge has been met in vitro with ‘six-lett...

The plausibility of any model in science comes from the extent of its interconnections to other models that are grounded in different premises and reasoning. Focusing research on paradoxes in those models, logic whereby they appear to generate unacceptable conclusions from seemingly indisputable premises, helps find those interconnections.

It has been four decades since formamide was first suggested to perform roles as a precursor and/or a solvent in prebiotic chemistry. However, little work has sought to integrate formamide into larger prebiotic schemes that might create prebiotic RNA, often proposed to have been the first Darwinian biopolymer. Here, we report that formamide can be...

According to the iconic model, the Watson-Crick double helix exploits nucleobase pairs that are both size complementary (big purines pair with small pyrimidines) and hydrogen bond complementary (hydrogen bond donors pair with hydrogen bond acceptors). Using a synthetic biology strategy, we report here the discovery of two new DNA-like systems that...

'Grand Challenges' offer ways to discover flaws in existing theory without first needing to guess what those flaws are. Our grand challenge here is to reproduce the Darwinism of terran biology, but on molecular platforms different from standard DNA. Access to Darwinism distinguishes the living from the non-living state. However, theory suggests tha...

A common criticism of “prebiotic chemistry research” is that it is done with starting materials that are too pure, in experiments that are too directed, to get results that are too scripted, under conditions that could never have existed on Earth. Planetary scientists in particular remark that these experiments often arise simply because a chemist...

Previously (Glushakova et al. 2017), a cellulose-based cationic (Q) paper derivatized with quaternary ammonium groups was shown to be a convenient platform to collect, preserve, and store nucleic acids (NAs) derived from mosquito vectors infected with pathogens for surveillance. NAs bind electrostatically to Q-paper, but the quantity of NA bound de...

The next challenge in synthetic biology is to be able to replicate synthetic nucleic acid sequences efficiently. The synthetic pair, 2-amino-8-(1-beta-d-2'- deoxyribofuranosyl) imidazo [1,2-a]-1,3,5-triazin-[8H]-4-one (trivially designated P) with 6-amino-3-(2'-deoxyribofuranosyl)-5-nitro-1H-pyridin-2-one (trivially designated Z), is replicated by...

Described here are the synthesis, enzymology and some applications of a purine nucleoside analog (H) designed to have two tautomeric forms, one complementary to thymidine (T), the other complementary to cytidine (C). The performance of H is compared by various metrics to performances of other 'biversal' analogs that similarly rely on tautomerism to...

Directed evolution was first applied to diverse libraries of DNA and RNA molecules a quarter century ago in the hope of gaining technology that would allow the creation of receptors, ligands, and catalysts on demand. Despite isolated successes, the outputs of this technology have been somewhat disappointing, perhaps because the four building blocks...

Cambridge Core - Computational Science and Modelling - Planets and Life - edited by Woodruff T. Sullivan, III

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different downstream patient-to-patient transmission potentials, and require very different patient treatments. Thus, recent Zika outbreaks make it urgent to develop tools that rapidly discriminate these viruses in...

Antibody fragments such as Fabs possess properties that can enhance protein and RNA crystallization and therefore can facilitate macromolecular structure determination. In particular, Fab BL3-6 binds to an AAACA RNA pentaloop closed by a GC pair with ∼100 nM affinity. The Fab and hairpin have served as a portable module for RNA crystallization. The...

The next challenge in synthetic biology is to be able to replicate synthetic nucleic acid sequences efficiently. The synthetic pair, 2-amino-8-(1-beta-D-2-deoxyribofuranosyl) imidazo [1,2-a]-1,3,5-triazin-[8H]-4-one (trivially designated P) with 6-amino-3-(2-deoxyribofuranosyl)-5-nitro-1H-pyridin-2-one (trivially designated Z), is replicated by cer...

Under the "RNA World" hypothesis, an early episode of natural history on Earth used RNA as the only genetically encoded molecule to catalyze steps in its metabolism catalysis. This, according to the hypothesis, included RNA catalysts that used RNA cofactors. However, the RNA World hypothesis places special demands on prebiotic chemistry, which must...

Significance Much recent research into the origins of life focuses on the hypothesis that RNA emerged on early Earth by an abiotic process, and gave Earth its first access to Darwinian evolution. This article provides a key step in this process. Here, we show that the phosphorylated ribonucleoside building blocks for RNA can be made stereoselective...

A goal of synthetic biology is to develop new nucleobases that retain the desirable properties of natural nucleobases at the same time as expanding the genetic alphabet. The non-standard Watson-Crick pair between imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione (X) and 2,4-diaminopyrimidine (K) does exactly this, pairing via complementary arrangemen...

Background Zika, dengue, and chikungunya are three mosquito-borne viruses having overlapping transmission vectors. They cause diseases having similar symptoms in human patients, but requiring different immediate management steps. Therefore, rapid (< one hour) discrimination of these three viruses in patient samples and trapped mosquitoes is needed....