Molecular Requirements of High-Fidelity Replication-Competent DNA Backbones for Orthogonal Chemical Ligation.

Arun Shivalingam,Tom Brown,Agnes E. S. Tyburn,Afaf H. El-Sagheer

doi:10.1021/jacs.6b11530

Arun Shivalingam, Tom Brown + Show 2 more

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https://doi.org/10.1021/jacs.6b11530

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Abstract

The molecular properties of the phosphodiester backbone that made it the evolutionary choice for the enzymatic replication of genetic information are not well understood. To address this, and to develop new chemical ligation strategies for assembly of biocompatible modified DNA, we have synthesized oligonucleotides containing several structurally and electronically varied artificial linkages. This has yielded a new highly promising ligation method based on amide backbone formation that is chemically orthogonal to CuAAC "click" ligation. A study of kinetics and fidelity of replication through these artificial linkages by primer extension, PCR, and deep sequencing reveals that a subtle interplay between backbone flexibility, steric factors, and ability to hydrogen bond to the polymerase modulates rapid and accurate information decoding. Even minor phosphorothioate modifications can impair the copying process, yet some radical triazole and amide DNA backbones perform surprisingly well, indicating that the phosphate group is not essential. These findings have implications in the field of synthetic biology.

Highlights

Over evolutionary time, the molecular structure of DNA has become intricately linked with the enzymatic tools that propagate it
Phosphate was abundant on prebiotic earth,[10] it has good reaction buffering capacity, it can act as a catalyst,[11] and it forms a stable phosphodiester linkage in DNA
Few studies have focused on replication or transcription through artificial DNA backbones, despite many analogues being synthesized for therapeutic applications.[12−18] Some basic information is available: minor phosphodiester modifications are accepted by polymerases,[19−21] an amide variant was imperfectly bypassed in primer−template experiments,[22] certain triazole-based backbones are tolerated by polymerases in vitro,[23−25] and one is even functional in bacterial and mammalian cells.[26,27]

Summary

Introduction

The molecular structure of DNA has become intricately linked with the enzymatic tools that propagate it. The order of slowest to fastest time-dependent backbone readthrough remained consistent with that of Taq. there was a lower local base pair bias, and Phusion generally replicated artificial linkages more efficiently than Taq at lower extension times.

Results

Conclusion