An allylic/acyclic adenosine nucleoside triphosphate for termination of DNA synthesis by DNA template-dependent polymerases

Abstract

An allylic adenosine triphosphate analog (AATP) was tested as a substrate for commercially available DNA polymerases. All but one of the enzymes assayed incorporated AATP opposite thymidine (T) with concomitant termination of the elongation reaction. A concentration of only 1 microM was sufficient for complete termination of the polymerization reaction for a short template mediated by Ampli Taq DNA polymerase FS (Taq FS). This result suggests that AATP could be used as a 2',3'-dideoxyadenosine-5'-triphosphate (ddA) surrogate. Kinetics of incorporation revealed that AATP was 48 times less efficiently incorporated than ddA. Furthermore, AATP was used in dye-primer sequencing as a substitute for ddA.

Enzymatic Incorporation in DNA of 1,5-Anhydrohexitol Nucleotides

“…Especially analogues altered in the 3′-position (32)(33)(34)37), 2′-position (35,36,38), or both (35) have been studied. But also acyclic analogues (49,50) and analogues with modifications in the 4′-position of the ring (51) were the subject of incorporation assays. Results from these experiments support a hypothesis that modified nucleosides and nucleotides can be accepted on the conditions that they possess a partly flattened sugar residue of limited flexibility (52).…”

Artificial nucleotide codons for enzymatic DNA synthesis

“…Moreover, this chemoenzymatic approach has a great potential to be further extended to longer lengths of shortmers (3-10 nt). We believe that this approach could be useful for the preparation of long XNA sequences that are accessible only with engineered polymerases, for the incorporation of nucleotides that are not compatible with polymerases or with unstable triphosphates (such as phosphorodiamidate morpholino (PMO) 48,49 or peptide nucleic acids (PNA) 50 ), and for the inclusion of multiple base-modifications in SELEX for the selection of chemically modified functional nucleic acids. 20,51…”

Artificial nucleotide codons for enzymatic DNA synthesis

“…Moreover, this approach has a great potential to be further extended to longer lengths of shortmers (3-10 nt). We believe that this approach could be useful for the preparation of long XNA sequences that are accessible only with engineered polymerases, for the incorporation of nucleotides that are not compatible with polymerases or with unstable triphosphates (such as phosphorodiamidate morpholino (PMO) 43,44 or peptide nucleic acids (PNA) 45 ), for the synthesis of oligonucleotides with stereo-controlled PS bonds, 46 and for the inclusion of base-modifications in SELEX for the selection of chemically modified functional nucleic acids. 17,27 The complexity of synthesizing all sequence variants (4 4 = 64 possibilities) that might be required for such practical applications can be partially alleviated by applying existing synthetic methods.…”