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David R Liu - One of the best experts on this subject based on the ideXlab platform.
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Translation of DNA into a Library of 13 000 Synthetic Small-Molecule Macrocycles Suitable for in Vitro Selection
2015Co-Authors: Brian N Tse, Thomas M Snyder, Yinghua Shen, David R LiuAbstract:DNA-templated organic synthesis enables the translation, Selection, and amplification of DNA sequences encoding synthetic small-molecule libraries. Previously we described the DNA-templated multistep synthesis and model in Vitro Selection of a pilot library of 65 macrocycles. in this work, we report several key developments that enable the DNA-templated synthesis of much larger (>10 000-membered) small-molecule libraries. We developed and validated a capping-based approach to DNA-templated library synthesis that increases final product yields, simplifies the structure and preparation of reagents, and reduces the number of required manipulations. To expand the size and structural diversity of the macrocycle library, we augmented the number of building blocks in each DNA-templated step from 4 to 12, selected 8 different starting scaffolds which result in 4 macrocycle ring sizes and 2 building-block orientations, and confirmed the ability of the 36 building blocks and 8 scaffolds to generate DNA-templated macrocycle products. We computationally generated and experimentally validated an expanded set of codons sufficient to support 1728 combinations of step 1, step 2, and step 3 building blocks. Finally, we developed new high-resolution LC/MS analysis methods to assess the quality of large DNA-templated small-molecule libraries. integrating these four developments, we executed the translation of 13 824 DNA templates into their corresponding small-molecule macrocycles. Analysis of the resulting libraries is consistent with excellent (>90%) representation of desired macrocycle products and a stringent test of sequence specificity suggests a high degree of sequence fidelity during translation. The quality and structural diversity of this expanded DNA-templated library provides a rich starting point for the discovery of functional synthetic small-molecule macrocycles
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in Vitro Selection of a dna templated small molecule library reveals a class of macrocyclic kinase inhibitors
Journal of the American Chemical Society, 2010Co-Authors: Ralph E Kleiner, Kaori Sakurai, Christoph E Dumelin, Gerald C Tiu, David R LiuAbstract:DNA-templated organic synthesis enables the translation of DNA sequences into synthetic small-molecule libraries suitable for in Vitro Selection. Previously, we described the DNA-templated multistep synthesis of a 13 824-membered small-molecule macrocycle library. Here, we report the discovery of small molecules that modulate the activity of kinase enzymes through the in Vitro Selection of this DNA-templated small-molecule macrocycle library against 36 biomedically relevant protein targets. DNA encoding Selection survivors was amplified by PCR and identified by ultra-high-throughput DNA sequencing. Macrocycles corresponding to DNA sequences enriched upon Selection against several protein kinases were synthesized on a multimilligram scale. in Vitro assays revealed that these macrocycles inhibit (or activate) the kinases against which they were selected with IC50 values as low as 680 nM. We characterized in depth a family of macrocycles enriched upon Selection against Src kinase, and showed that inhibition ...
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translation of dna into a library of 13 000 synthetic small molecule macrocycles suitable for in Vitro Selection
Journal of the American Chemical Society, 2008Co-Authors: Brian N Tse, Thomas M Snyder, Yinghua Shen, David R LiuAbstract:DNA-templated organic synthesis enables the translation, Selection, and amplification of DNA sequences encoding synthetic small-molecule libraries. Previously we described the DNA-templated multistep synthesis and model in Vitro Selection of a pilot library of 65 macrocycles. in this work, we report several key developments that enable the DNA-templated synthesis of much larger (>10 000-membered) small-molecule libraries. We developed and validated a capping-based approach to DNA-templated library synthesis that increases final product yields, simplifies the structure and preparation of reagents, and reduces the number of required manipulations. To expand the size and structural diversity of the macrocycle library, we augmented the number of building blocks in each DNA-templated step from 4 to 12, selected 8 different starting scaffolds which result in 4 macrocycle ring sizes and 2 building-block orientations, and confirmed the ability of the 36 building blocks and 8 scaffolds to generate DNA-templated ma...
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translation of dna into a library of 13 000 synthetic small molecule macrocycles suitable for in Vitro Selection
Journal of the American Chemical Society, 2008Co-Authors: Brian N Tse, Thomas M Snyder, Yinghua Shen, David R LiuAbstract:DNA-templated organic synthesis enables the translation, Selection, and amplification of DNA sequences encoding synthetic small-molecule libraries. Previously we described the DNA-templated multistep synthesis and model in Vitro Selection of a pilot library of 65 macrocycles. in this work, we report several key developments that enable the DNA-templated synthesis of much larger (>10,000-membered) small-molecule libraries. We developed and validated a capping-based approach to DNA-templated library synthesis that increases final product yields, simplifies the structure and preparation of reagents, and reduces the number of required manipulations. To expand the size and structural diversity of the macrocycle library, we augmented the number of building blocks in each DNA-templated step from 4 to 12, selected 8 different starting scaffolds which result in 4 macrocycle ring sizes and 2 building-block orientations, and confirmed the ability of the 36 building blocks and 8 scaffolds to generate DNA-templated macrocycle products. We computationally generated and experimentally validated an expanded set of codons sufficient to support 1728 combinations of step 1, step 2, and step 3 building blocks. Finally, we developed new high-resolution LC/MS analysis methods to assess the quality of large DNA-templated small-molecule libraries. integrating these four developments, we executed the translation of 13,824 DNA templates into their corresponding small-molecule macrocycles. Analysis of the resulting libraries is consistent with excellent (>90%) representation of desired macrocycle products and a stringent test of sequence specificity suggests a high degree of sequence fidelity during translation. The quality and structural diversity of this expanded DNA-templated library provides a rich starting point for the discovery of functional synthetic small-molecule macrocycles.
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reaction discovery enabled by dna templated synthesis and in Vitro Selection
Nature, 2004Co-Authors: Matthew W Kanan, Mary M Rozenman, Kaori Sakurai, Thomas M Snyder, David R LiuAbstract:Current approaches to reaction discovery focus on one particular transformation. Typically, researchers choose substrates based on their predicted ability to serve as precursors for the target structure, then evaluate reaction conditions1,2,3,4,5,6 for their ability to effect product formation. This approach is ideal for addressing specific reactivity problems, but its focused nature might leave many areas of chemical reactivity unexplored. Here we report a reaction discovery approach that uses DNA-templated organic synthesis7,8,9,10 and in Vitro Selection to simultaneously evaluate many combinations of different substrates for bond-forming reactions in a single solution. Watson–Crick base pairing controls the effective molarities of substrates tethered to DNA strands; bond-forming substrate combinations are then revealed using in Vitro Selection for bond formation, PCR amplification and DNA microarray analysis. Using this approach, we discovered an efficient and mild carbon–carbon bond-forming reaction that generates an enone from an alkyne and alkene using an inorganic palladium catalyst. Although this approach is restricted to conditions and catalysts that are at least partially compatible with DNA, we expect that its versatility and efficiency will enable the discovery of additional reactions between a wide range of substrates.
Brian N Tse - One of the best experts on this subject based on the ideXlab platform.
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Translation of DNA into a Library of 13 000 Synthetic Small-Molecule Macrocycles Suitable for in Vitro Selection
2015Co-Authors: Brian N Tse, Thomas M Snyder, Yinghua Shen, David R LiuAbstract:DNA-templated organic synthesis enables the translation, Selection, and amplification of DNA sequences encoding synthetic small-molecule libraries. Previously we described the DNA-templated multistep synthesis and model in Vitro Selection of a pilot library of 65 macrocycles. in this work, we report several key developments that enable the DNA-templated synthesis of much larger (>10 000-membered) small-molecule libraries. We developed and validated a capping-based approach to DNA-templated library synthesis that increases final product yields, simplifies the structure and preparation of reagents, and reduces the number of required manipulations. To expand the size and structural diversity of the macrocycle library, we augmented the number of building blocks in each DNA-templated step from 4 to 12, selected 8 different starting scaffolds which result in 4 macrocycle ring sizes and 2 building-block orientations, and confirmed the ability of the 36 building blocks and 8 scaffolds to generate DNA-templated macrocycle products. We computationally generated and experimentally validated an expanded set of codons sufficient to support 1728 combinations of step 1, step 2, and step 3 building blocks. Finally, we developed new high-resolution LC/MS analysis methods to assess the quality of large DNA-templated small-molecule libraries. integrating these four developments, we executed the translation of 13 824 DNA templates into their corresponding small-molecule macrocycles. Analysis of the resulting libraries is consistent with excellent (>90%) representation of desired macrocycle products and a stringent test of sequence specificity suggests a high degree of sequence fidelity during translation. The quality and structural diversity of this expanded DNA-templated library provides a rich starting point for the discovery of functional synthetic small-molecule macrocycles
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translation of dna into a library of 13 000 synthetic small molecule macrocycles suitable for in Vitro Selection
Journal of the American Chemical Society, 2008Co-Authors: Brian N Tse, Thomas M Snyder, Yinghua Shen, David R LiuAbstract:DNA-templated organic synthesis enables the translation, Selection, and amplification of DNA sequences encoding synthetic small-molecule libraries. Previously we described the DNA-templated multistep synthesis and model in Vitro Selection of a pilot library of 65 macrocycles. in this work, we report several key developments that enable the DNA-templated synthesis of much larger (>10 000-membered) small-molecule libraries. We developed and validated a capping-based approach to DNA-templated library synthesis that increases final product yields, simplifies the structure and preparation of reagents, and reduces the number of required manipulations. To expand the size and structural diversity of the macrocycle library, we augmented the number of building blocks in each DNA-templated step from 4 to 12, selected 8 different starting scaffolds which result in 4 macrocycle ring sizes and 2 building-block orientations, and confirmed the ability of the 36 building blocks and 8 scaffolds to generate DNA-templated ma...
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translation of dna into a library of 13 000 synthetic small molecule macrocycles suitable for in Vitro Selection
Journal of the American Chemical Society, 2008Co-Authors: Brian N Tse, Thomas M Snyder, Yinghua Shen, David R LiuAbstract:DNA-templated organic synthesis enables the translation, Selection, and amplification of DNA sequences encoding synthetic small-molecule libraries. Previously we described the DNA-templated multistep synthesis and model in Vitro Selection of a pilot library of 65 macrocycles. in this work, we report several key developments that enable the DNA-templated synthesis of much larger (>10,000-membered) small-molecule libraries. We developed and validated a capping-based approach to DNA-templated library synthesis that increases final product yields, simplifies the structure and preparation of reagents, and reduces the number of required manipulations. To expand the size and structural diversity of the macrocycle library, we augmented the number of building blocks in each DNA-templated step from 4 to 12, selected 8 different starting scaffolds which result in 4 macrocycle ring sizes and 2 building-block orientations, and confirmed the ability of the 36 building blocks and 8 scaffolds to generate DNA-templated macrocycle products. We computationally generated and experimentally validated an expanded set of codons sufficient to support 1728 combinations of step 1, step 2, and step 3 building blocks. Finally, we developed new high-resolution LC/MS analysis methods to assess the quality of large DNA-templated small-molecule libraries. integrating these four developments, we executed the translation of 13,824 DNA templates into their corresponding small-molecule macrocycles. Analysis of the resulting libraries is consistent with excellent (>90%) representation of desired macrocycle products and a stringent test of sequence specificity suggests a high degree of sequence fidelity during translation. The quality and structural diversity of this expanded DNA-templated library provides a rich starting point for the discovery of functional synthetic small-molecule macrocycles.
Thomas M Snyder - One of the best experts on this subject based on the ideXlab platform.
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Translation of DNA into a Library of 13 000 Synthetic Small-Molecule Macrocycles Suitable for in Vitro Selection
2015Co-Authors: Brian N Tse, Thomas M Snyder, Yinghua Shen, David R LiuAbstract:DNA-templated organic synthesis enables the translation, Selection, and amplification of DNA sequences encoding synthetic small-molecule libraries. Previously we described the DNA-templated multistep synthesis and model in Vitro Selection of a pilot library of 65 macrocycles. in this work, we report several key developments that enable the DNA-templated synthesis of much larger (>10 000-membered) small-molecule libraries. We developed and validated a capping-based approach to DNA-templated library synthesis that increases final product yields, simplifies the structure and preparation of reagents, and reduces the number of required manipulations. To expand the size and structural diversity of the macrocycle library, we augmented the number of building blocks in each DNA-templated step from 4 to 12, selected 8 different starting scaffolds which result in 4 macrocycle ring sizes and 2 building-block orientations, and confirmed the ability of the 36 building blocks and 8 scaffolds to generate DNA-templated macrocycle products. We computationally generated and experimentally validated an expanded set of codons sufficient to support 1728 combinations of step 1, step 2, and step 3 building blocks. Finally, we developed new high-resolution LC/MS analysis methods to assess the quality of large DNA-templated small-molecule libraries. integrating these four developments, we executed the translation of 13 824 DNA templates into their corresponding small-molecule macrocycles. Analysis of the resulting libraries is consistent with excellent (>90%) representation of desired macrocycle products and a stringent test of sequence specificity suggests a high degree of sequence fidelity during translation. The quality and structural diversity of this expanded DNA-templated library provides a rich starting point for the discovery of functional synthetic small-molecule macrocycles
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translation of dna into a library of 13 000 synthetic small molecule macrocycles suitable for in Vitro Selection
Journal of the American Chemical Society, 2008Co-Authors: Brian N Tse, Thomas M Snyder, Yinghua Shen, David R LiuAbstract:DNA-templated organic synthesis enables the translation, Selection, and amplification of DNA sequences encoding synthetic small-molecule libraries. Previously we described the DNA-templated multistep synthesis and model in Vitro Selection of a pilot library of 65 macrocycles. in this work, we report several key developments that enable the DNA-templated synthesis of much larger (>10,000-membered) small-molecule libraries. We developed and validated a capping-based approach to DNA-templated library synthesis that increases final product yields, simplifies the structure and preparation of reagents, and reduces the number of required manipulations. To expand the size and structural diversity of the macrocycle library, we augmented the number of building blocks in each DNA-templated step from 4 to 12, selected 8 different starting scaffolds which result in 4 macrocycle ring sizes and 2 building-block orientations, and confirmed the ability of the 36 building blocks and 8 scaffolds to generate DNA-templated macrocycle products. We computationally generated and experimentally validated an expanded set of codons sufficient to support 1728 combinations of step 1, step 2, and step 3 building blocks. Finally, we developed new high-resolution LC/MS analysis methods to assess the quality of large DNA-templated small-molecule libraries. integrating these four developments, we executed the translation of 13,824 DNA templates into their corresponding small-molecule macrocycles. Analysis of the resulting libraries is consistent with excellent (>90%) representation of desired macrocycle products and a stringent test of sequence specificity suggests a high degree of sequence fidelity during translation. The quality and structural diversity of this expanded DNA-templated library provides a rich starting point for the discovery of functional synthetic small-molecule macrocycles.
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translation of dna into a library of 13 000 synthetic small molecule macrocycles suitable for in Vitro Selection
Journal of the American Chemical Society, 2008Co-Authors: Brian N Tse, Thomas M Snyder, Yinghua Shen, David R LiuAbstract:DNA-templated organic synthesis enables the translation, Selection, and amplification of DNA sequences encoding synthetic small-molecule libraries. Previously we described the DNA-templated multistep synthesis and model in Vitro Selection of a pilot library of 65 macrocycles. in this work, we report several key developments that enable the DNA-templated synthesis of much larger (>10 000-membered) small-molecule libraries. We developed and validated a capping-based approach to DNA-templated library synthesis that increases final product yields, simplifies the structure and preparation of reagents, and reduces the number of required manipulations. To expand the size and structural diversity of the macrocycle library, we augmented the number of building blocks in each DNA-templated step from 4 to 12, selected 8 different starting scaffolds which result in 4 macrocycle ring sizes and 2 building-block orientations, and confirmed the ability of the 36 building blocks and 8 scaffolds to generate DNA-templated ma...
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reaction discovery enabled by dna templated synthesis and in Vitro Selection
Nature, 2004Co-Authors: Matthew W Kanan, Mary M Rozenman, Kaori Sakurai, Thomas M Snyder, David R LiuAbstract:Current approaches to reaction discovery focus on one particular transformation. Typically, researchers choose substrates based on their predicted ability to serve as precursors for the target structure, then evaluate reaction conditions1,2,3,4,5,6 for their ability to effect product formation. This approach is ideal for addressing specific reactivity problems, but its focused nature might leave many areas of chemical reactivity unexplored. Here we report a reaction discovery approach that uses DNA-templated organic synthesis7,8,9,10 and in Vitro Selection to simultaneously evaluate many combinations of different substrates for bond-forming reactions in a single solution. Watson–Crick base pairing controls the effective molarities of substrates tethered to DNA strands; bond-forming substrate combinations are then revealed using in Vitro Selection for bond formation, PCR amplification and DNA microarray analysis. Using this approach, we discovered an efficient and mild carbon–carbon bond-forming reaction that generates an enone from an alkyne and alkene using an inorganic palladium catalyst. Although this approach is restricted to conditions and catalysts that are at least partially compatible with DNA, we expect that its versatility and efficiency will enable the discovery of additional reactions between a wide range of substrates.
Larry Gold - One of the best experts on this subject based on the ideXlab platform.
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high affinity ligands from in Vitro Selection complex targets
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Kevin N Morris, Kirk B. Jensen, Carol M Julin, Michael D Weil, Larry GoldAbstract:Human red blood cell membranes were used as a model system to determine if the systematic evolution of ligands by exponential enrichment (SELEX) methodology, an in Vitro protocol for isolating high-affinity oligonucleotides that bind specifically to virtually any single protein, could be used with a complex mixture of potential targets. Ligands to multiple targets were generated simultaneously during the Selection process, and the binding affinities of these ligands for their targets are comparable to those found in similar experiments against pure targets. A secondary Selection scheme, deconvolution-SELEX, facilitates rapid isolation of the ligands to targets of special interest within the mixture. SELEX provides high-affinity compounds for multiple targets in a mixture and might allow a means for dissecting complex biological systems.
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using in Vitro Selection to direct the covalent attachment of human immunodeficiency virus type 1 rev protein to high affinity rna ligands
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Kirk B. Jensen, Brent Atkinson, Michael Willis, Tad H Koch, Larry GoldAbstract:Abstract We have used an in Vitro Selection procedure called crosslinking SELEX (SELEX = systematic evolution of ligands by exponential enrichment) to identify RNA sequences that bind with high affinity and crosslink to the Rev protein from human immunodeficiency virus type 1 (HIV-1). A randomized RNA library substituted with the photoreactive chromophore 5-iodouracil was irradiated with monochromatic UV light in the presence of Rev. Those sequences with the ability to photocrosslink to Rev were partitioned from the rest of the RNA pool, amplified, and used for the next round of Selection. Rounds of photocrosslinking Selection were alternated with rounds of Selection for RNA sequences with high affinity to Rev. This iterative, dual-Selection method yielded RNA molecules with subnanomolar dissociation constants and high efficiency photocrosslinking to Rev. Some of the RNA molecules isolated by this procedure form a stable complex with Rev that is resistant to denaturing gel electrophoresis in the absence of UV irradiation. in Vitro Selection of nucleic acids by using modified nucleotides allows the isolation of nucleic acid molecules with potentially limitless chemical capacities to covalently attack a target molecule.
Jack W Szostak - One of the best experts on this subject based on the ideXlab platform.
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in Vitro Selection of ribozyme ligases that use prebiotically plausible 2-aminoimidazole-activated substrates.
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Travis Walton, Saurja Dasgupta, Daniel Duzdevich, Jack W SzostakAbstract:The hypothesized central role of RNA in the origin of life suggests that RNA propagation predated the advent of complex protein enzymes. A critical step of RNA replication is the template-directed synthesis of a complementary strand. Two experimental approaches have been extensively explored in the pursuit of demonstrating protein-free RNA synthesis: template-directed nonenzymatic RNA polymerization using intrinsically reactive monomers and ribozyme-catalyzed polymerization using more stable substrates such as biological 5′-triphosphates. Despite significant progress in both approaches in recent years, the assembly and copying of functional RNA sequences under prebiotic conditions remains a challenge. Here, we explore an alternative approach to RNA-templated RNA copying that combines ribozyme catalysis with RNA substrates activated with a prebiotically plausible leaving group, 2-aminoimidazole (2AI). We applied in Vitro Selection to identify ligase ribozymes that catalyze phosphodiester bond formation between a template-bound primer and a phosphor-imidazolide–activated oligomer. Sequencing revealed the progressive enrichment of 10 abundant sequences from a random sequence pool. Ligase activity was detected in all 10 RNA sequences; all required activation of the ligator with 2AI and generated a 3′-5′ phosphodiester bond. We propose that ribozyme catalysis of phosphodiester bond formation using intrinsically reactive RNA substrates, such as imidazolides, could have been an evolutionary step connecting purely nonenzymatic to ribozyme-catalyzed RNA template copying during the origin of life.
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in Vitro Selection of functional lantipeptides
Journal of the American Chemical Society, 2012Co-Authors: Frank Hofmann, Jack W Szostak, Florian P SeebeckAbstract:in this report we present a method to identify functional artificial lantipeptides. in Vitro translation coupled with an enzyme-free protocol for posttranslational modification allows preparation of more than 1011 different lanthionine containing peptides. This diversity can be searched for functional molecules using mRNA-lantipeptide display. We validated this approach by isolating binders toward Sortase A, a transamidase which is required for virulence of Staphylococcus aureus. The interaction of selected lantipeptides with Sortase A is highly dependent on the presence of a (2S,6R)-lanthionine in the peptide and an active conformation of the protein.
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in Vitro Selection of highly modified cyclic peptides that act as tight binding inhibitors
Journal of the American Chemical Society, 2012Co-Authors: Yollete Guillen V Schlippe, Matthew C T Hartman, Kristopher Josephson, Jack W SzostakAbstract:There is a great demand for the discovery of new therapeutic molecules that combine the high specificity and affinity of biologic drugs with the bioavailability and lower cost of small molecules. Small, natural-product-like peptides hold great promise in bridging this gap; however, access to libraries of these compounds has been a limitation. Since ribosomal peptides may be subjected to in Vitro Selection techniques, the generation of extremely large libraries (>1013) of highly modified macrocyclic peptides may provide a powerful alternative for the generation and Selection of new useful bioactive molecules. Moreover, the incorporation of many non-proteinogenic amino acids into ribosomal peptides in conjunction with macrocyclization should enhance the drug-like features of these libraries. Here we show that mRNA-display, a technique that allows the in Vitro Selection of peptides, can be applied to the evolution of macrocyclic peptides that contain a majority of unnatural amino acids. We describe the isola...
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an in Vitro Selection system for tna
Journal of the American Chemical Society, 2005Co-Authors: Justin K Ichida, Keyong Zou, Allen Horhota, Larry W Mclaughlin, Jack W SzostakAbstract:(3‘-2‘)-α-l-Threose nucleic acid (TNA) is an unnatural polymer that possesses the rare ability to base-pair with RNA, DNA, and itself. This feature, coupled with its chemical simplicity, makes TNA of interest as a possible progenitor of RNA during the early history of life. To evaluate the functional potential of TNA, we have developed a system for the in Vitro Selection of TNA. We identified the Therminator DNA polymerase as a remarkably efficient DNA-dependent TNA polymerase capable of polymerizing more than 50 tNTPs. We have also developed a method of covalently linking a DNA template to the TNA strand that it encodes, thus obviating the need for a TNA-dependent DNA polymerase during cycles of Selection.
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in Vitro Selection of functional nucleic acids
Annual Review of Biochemistry, 1999Co-Authors: David S Wilson, Jack W SzostakAbstract:■ Abstract in Vitro Selection allows rare functional RNA or DNA molecules to be isolated from pools of over 10 15 different sequences. This approach has been used to identify RNA and DNA ligands for numerous small molecules, and recent threedimensional structure solutions have revealed the basis for ligand recognition in several cases. By selecting high-affinity and -specificity nucleic acid ligands for proteins, promising new therapeutic and diagnostic reagents have been identified. Selection experiments have also been carried out to identify ribozymes that catalyze a variety of chemical transformations, including RNA cleavage, ligation, and synthesis, as well as alkylation and acyl-transfer reactions and N-glycosidic and peptide bond formation. The existence of such RNA enzymes supports the notion that ribozymes could have directed a primitive metabolism before the evolution of protein synthesis. New in Vitro protein Selection techniques should allow for a direct comparison of the frequency of ligand binding and catalytic structures in pools of random sequence polynucleotides versus polypeptides.
