Structural Basis of the Mispairing of an Artificially Expanded Genetic Information System

Linus F Reichenbach,Ahmad Ahmad Sobri,Nathan R Zaccai,Christopher Agnew,Nicholas Burton,Lucy P Eperon,Sara De Ornellas,Ian C Eperon,R Leo Brady,Glenn A Burley

doi:10.1016/j.chempr.2016.11.009

Linus F Reichenbach, Ahmad Ahmad Sobri + Show 8 more

Open Access

https://doi.org/10.1016/j.chempr.2016.11.009

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Abstract

Summary Relative to naturally occurring Watson-Crick base pairs, the synthetic nucleotide P pairs with Z within DNA duplexes through a unique hydrogen-bond arrangement. The loss of this synthetic genetic information by PCR results in the conversion of P-Z into a G-C base pair. Here, we show structural and spectroscopic evidence that the loss of this synthetic genetic information occurs via G-Z mispairing. Remarkably, the G-Z mispair is both plastic and pH dependent; it forms a double-hydrogen-bonded slipped pair at pH 7.8 and a triple-hydrogen-bonded Z-G pair when the pH is above 7.8. This study highlights the need for robust structural and functional methods to elucidate the mechanisms of mutation in the development of next-generation synthetic genetic base pairs.

Highlights

Nucleic acids are the fundamental repository of genetic information found in living organisms
Using a combination of PCR analysis of base-pair fidelity, X-ray crystallography, UV-visible (UV-Vis) thermal melts, and circular dichroism (CD) spectroscopy, we show that G-Z mispairing is remarkably plastic; it pairs via a mixture of canonical and non-canonical regimes that are aligned with the pKa of the Z N1-H (Figure 1C).[22]
The archetypical Dickerson dodecamer d(CGCGAATTCGCG)[2] (ODN1), for which a crystal structure has previously been determined in the absence of host protein and presence of Ca2+ (PDB: 463D),[29] was used as a comparison for our structural and spectroscopic studies because this self-complementary sequence forms B-type DNA duplexes with both natural and modified nucleotides.[30,31,32,33,34]

Summary

Introduction

Nucleic acids are the fundamental repository of genetic information found in living organisms. The development of an expanded genetic repertoire might have many uses, but it would require the design of synthetic base pairs that pair orthogonally and exclusively with each other and not with naturally occurring nucleotides.[4,5,6,7,8,9,10,11] The Hirao and Romesberg groups have developed synthetic base pairs that rely on complementarity of shape rather than hydrogen bonding to pair preferentially with each other Both systems enable replication fidelities approaching that of natural Watson-Crick base pairs.[4,5,10,12,13,14,15,16,17,18,19] A particular mutational hallmark of these hydrophobic base pairs is the gradual conversion of the synthetic base pair into an A-T pair observed both in cellulo and via repeated rounds of PCR, the structural basis for the loss of synthetic information is at present not known.[16,18]

Results

Discussion

Conclusion