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Investigation of the DNA-dependent cyclohexenyl nucleic acid polymerization and the cyclohexenyl nucleic acid-dependent DNA polymerization.

ABSTRACT: DNA polymerases from different evolutionary families [Vent (exo-) DNA polymerase from the B-family polymerases, Taq DNA polymerase from the A-family polymerases and HIV reverse transcriptase from the reverse transcriptase family] were examined for their ability to incorporate the sugar-modified cyclohexenyl nucleoside triphosphates. All enzymes were able to use the cyclohexenyl nucleotides as a substrate. Using Vent (exo-) DNA polymerase and HIV reverse transcriptase, we were even able to incorporate seven consecutive cyclohexenyl nucleotides. Using a cyclohexenyl nucleic acid (CeNA) template, all enzymes tested were also able to synthesize a short DNA fragment. Since the DNA-dependent CeNA polymerization and the CeNA-dependent DNA polymerization is possible to a limited extend, we suggest CeNA as an ideal candidate to use in directed evolution methods for the development of a polymerase capable of replicating CeNA.

SUBMITTER: Kempeneers V

PROVIDER: S-EPMC1175020 | biostudies-literature | 2005

REPOSITORIES: biostudies-literature

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Investigation of the DNA-dependent cyclohexenyl nucleic acid polymerization and the cyclohexenyl nucleic acid-dependent DNA polymerization.

Kempeneers Veerle V Renders Marleen M Froeyen Matheus M Herdewijn Piet P

Nucleic acids research 20050712 12

DNA polymerases from different evolutionary families [Vent (exo-) DNA polymerase from the B-family polymerases, Taq DNA polymerase from the A-family polymerases and HIV reverse transcriptase from the reverse transcriptase family] were examined for their ability to incorporate the sugar-modified cyclohexenyl nucleoside triphosphates. All enzymes were able to use the cyclohexenyl nucleotides as a substrate. Using Vent (exo-) DNA polymerase and HIV reverse transcriptase, we were even able to incorp ...[more]

PMID: 16027107

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Project description:The DNA-templated polymerization of synthetic building blocks provides a potential route to the laboratory evolution of sequence-defined polymers with structures and properties not necessarily limited to those of natural biopolymers. We previously reported the efficient and sequence-specific DNA-templated polymerization of peptide nucleic acid (PNA) aldehydes. Here, we report the enzyme-free, DNA-templated polymerization of side-chain-functionalized PNA tetramer and pentamer aldehydes. We observed that polymerization of tetramer and pentamer PNA building blocks with a single lysine-based side chain at various positions in the building block could proceed efficiently and sequence specifically. In addition, DNA-templated polymerization also proceeded efficiently and in a sequence-specific manner with pentamer PNA aldehydes containing two or three lysine side chains in a single building block to generate more densely functionalized polymers. To further our understanding of side-chain compatibility and expand the capabilities of this system, we also examined the polymerization efficiencies of 20 pentamer building blocks each containing one of five different side-chain groups and four different side-chain regio- and stereochemistries. Polymerization reactions were efficient for all five different side-chain groups and for three of the four combinations of side-chain regio- and stereochemistries. Differences in the efficiency and initial rate of polymerization correlate with the apparent melting temperature of each building block, which is dependent on side-chain regio- and stereochemistry but relatively insensitive to side-chain structure among the substrates tested. Our findings represent a significant step toward the evolution of sequence-defined synthetic polymers and also demonstrate that enzyme-free nucleic acid-templated polymerization can occur efficiently using substrates with a wide range of side-chain structures, functionalization positions within each building block, and functionalization densities.

Project description:We report the design and synthesis of 2'-fluoro cyclohexenyl nucleic acid (F-CeNA) pyrimidine phosphoramidites and the synthesis and biophysical, structural, and biological evaluation of modified oligonucleotides. The synthesis of the nucleoside phosphoramidites was accomplished in multigram quantities starting from commercially available methyl-D-mannose pyranoside. Installation of the fluorine atom was accomplished using nonafluorobutanesulfonyl fluoride, and the cyclohexenyl ring system was assembled by means of a palladium-catalyzed Ferrier rearrangement. Installation of the nucleobase was carried out under Mitsunobu conditions followed by standard protecting group manipulations to provide the desired pyrimidine phosphoramidites. Biophysical evaluation indicated that F-CeNA shows behavior similar to that of a 2'-modified nucleotide, and duplexes with RNA showed slightly lower duplex thermostability as compared to that of the more rigid 3'-fluoro hexitol nucleic acid (FHNA). However, F-CeNA modified oligonucleotides were significantly more stable against digestion by snake venom phosphodiesterases (SVPD) as compared to unmodified DNA, 2'-fluoro RNA (FRNA), 2'-methoxyethyl RNA (MOE), and FHNA modified oligonucleotides. Examination of crystal structures of a modified DNA heptamer duplex d(GCG)-T*-d(GCG):d(CGCACGC) by X-ray crystallography indicated that the cyclohexenyl ring system exhibits both the (3)H(2) and (2)H(3) conformations, similar to the C3'-endo/C2'-endo conformation equilibrium seen in natural furanose nucleosides. In the (2)H(3) conformation, the equatorial fluorine engages in a relatively close contact with C8 (2.94 Å) of the 3'-adjacent dG nucleotide that may represent a pseudo hydrogen bond. In contrast, the cyclohexenyl ring of F-CeNA was found to exist exclusively in the (3)H(2) (C3'-endo like) conformation in the crystal structure of the modified A-form DNA decamer duplex [d(GCGTA)-T*-d(ACGC)](2.) In an animal experiment, a 16-mer F-CeNA gapmer ASO showed similar RNA affinity but significantly improved activity compared to that of a sequence matched MOE ASO, thus establishing F-CeNA as a useful modification for antisense applications.

Dataset Information

Investigation of the DNA-dependent cyclohexenyl nucleic acid polymerization and the cyclohexenyl nucleic acid-dependent DNA polymerization.

Publications

Investigation of the DNA-dependent cyclohexenyl nucleic acid polymerization and the cyclohexenyl nucleic acid-dependent DNA polymerization.

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