Templated synthesis of nylon nucleic acids and characterization by nuclease digestion

Abstract

Nylon nucleic acids containing oligouridine nucleotides with pendent polyamide linkers and flanked by unmodified heteronucleotide sequences were prepared by DNA templated synthesis. Templation was more efficient than the single-stranded synthesis: Coupling step yields were as high as 99.2%, with up to 7 amide linkages formed in the synthesis of a molecule containing 8 modified nucleotides. Controlled digestion by calf spleen phosphodiesterase enabled the mapping of modified nucleotides in the sequences. A combination of complete degradation of nylon nucleic acids by snake venom phosphodiesterase and dephosphorylation of the resulting nucleotide fragments by bacterial alkaline phosphatase, followed by LCMS analysis, clarified the linear structure of the oligo-amide linkages. The templated synthesis strategy afforded nylon nucleic acids in the target structure and was compatible with the presence heteronucleotides. The complete digestion procedure produced a new species of DNA analogues, nylon ribonucleosides, which display nucleosides attached via a 2′-alkylthio linkage to each diamine and dicarboxylate repeat unit of the original nylon nucleic acids. The binding affinity of a nylon ribonucleoside octamer to the complementary DNA was evaluated by thermal denaturing experiments. The octamer was found to form stable duplexes with an inverse dependence on salt concentration, in contrast to the salt-dependent DNA control.

Nuclease-Resistant DNA via High-Density Packing in Polymeric Micellar Nanoparticle Coronas

“…Therefore, we subjected the single-stranded particles and corresponding ssDNA analogues to Snake Venom Phosphodiesterase (Phosphodiesterase I from Crotalus adamanteus ), an enzyme known for its aggressive 3′-exonuclease activity and routinely utilized for complete digestion of synthetic oligonucleotides. 22,23 …”

“…In addition, we sought to answer whether DPA nanoparticles serve to protect DNA against general exonuclease digestion and not just specifically Exo III digestion. Therefore, we subjected the single-stranded particles and corresponding ssDNA analogues to snake-venom phosphodiesterase (phosphodiesterase I from Crotalus adamanteus), an enzyme known for its aggressive 3′-exonuclease activity and routinely utilized for complete digestion of synthetic oligonucleotides. , …”

Towards XNA nanotechnology: new materials from synthetic genetic polymers

“…For instance, if the nanostructure falsework is introduced as RNA, the need for toeholds may even be circumvented by the use of enzymes that selectively degrade RNA over DNA. The principle of DNA falsework has been successfully used to direct chemical synthesis of short sequence-defined oligomeric compounds [76,77] .…”

Site-specific inter-strand cross-links of DNA duplexes

“…26, 27, 28, 29 In these reports, we incorporated special nucleotides into DNA molecules and joined the derivatized nucleotides together by forming amide bonds in a reaction using the chemical coupling reagent DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) in aqueous solution. We have also used this methodology to ligate DNA strands by 2′,2′ coupling.…”

“…Previously, we reported the addition of 2 0 -alkylamine or 2 0alkylcarboxylate groups to nucleotides where the 2 0 -oxygen of a ribonucleotide had been replaced by a sulfur atom. [26][27][28][29] In these reports, we incorporated special nucleotides into DNA molecules and joined the derivatized nucleotides together by forming amide bonds in a reaction using the chemical coupling reagent DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) in aqueous solution. We have also used this methodology to ligate DNA strands by 2 0 ,2 0 coupling.…”