The Structural Basis for Processing of Unnatural Base Pairs by DNA Polymerases

Abstract

Unnatural base pairs (UBPs) greatly increase the diversity of DNA and RNA, furthering their broad range of molecular biological and biotechnological approaches. Different candidates have been developed whereby alternative hydrogen‐bonding patterns and hydrophobic and packing interactions have turned out to be the most promising base‐pairing concepts to date. The key in many applications is the highly efficient and selective acceptance of artificial base pairs by DNA polymerases, which enables amplification of the modified DNA. In this Review, computational as well as experimental studies that were performed to characterize the pairing behavior of UBPs in free duplex DNA or bound to the active site of KlenTaq DNA polymerase are highlighted. The structural studies, on the one hand, elucidate how base pairs lacking hydrogen bonds are accepted by these enzymes and, on the other hand, highlight the influence of one or several consecutive UBPs on the structure of a DNA double helix. Understanding these concepts facilitates optimization of future UBPs for the manifold fields of applications.

Computational investigations of polymerase enzymes: Structure, function, inhibition, and biotechnology

“…The expansion of the genetic alphabet, through the creation of UBPs from modified nucleotides, enables the storage of additional information in DNA. 209,210 Furthermore, the increased chemical and structural diversity of the modified DNA and RNA would broaden their molecular biological and biotechnological applications to, for example, production of aptamers that bind to proteins and cells, incorporation of unnatural amino acids into proteins, and generation of semisynthetic organisms. However, the major challenge is to identify or develop DNA Pols that can incorporate UBPs with high catalytic efficiency and fidelity and maintain high extension efficiency after the incorporation of the UBP.…”

Enzymatic synthesis of biphenyl-DNA oligonucleotides

“…Collectively, these results demonstrate the possibility of using polymerases to synthesize dBph-containing oligonucleotides and could be expanded to other C-nucleotides. This approach can benefit from the use of engineered polymerases [8,78,79] and work towards this aim is currently under way in our laboratory.…”

Enzymatic Construction of Artificial Base Pairs: The Effect of Metal Shielding

“…In the general context of identifying novel UBPs, the nucleotide candidate needs to be fully orthogonal to canonical DNA and has to be inserted opposite a templating modified nucleotide with an error rate not lower than 10 À 3 . [39] These rather stringent criteria can be met if the UBP does not cause major perturbations of the major and minor groove interactions of the base pair under construction and the polymerase. Hence, various parameters need to be taken into consideration when designing UBPs including stacking capacity, [40] thermal stability of the base pair, solvent interactions, [41] pK a values of the respective nucleotides, [42] size-complementarity, [7b,43] and minor groove interactions.…”

“…This is particularly true for base‐modified nucleotide analogues into which a variety of functional groups ranging from small residues [36] to large fragments including polymerases [37] or even antibodies [38] could be introduced into nucleic acids by polymerases. In the general context of identifying novel UBPs, the nucleotide candidate needs to be fully orthogonal to canonical DNA and has to be inserted opposite a templating modified nucleotide with an error rate not lower than 10 −3 [39] . These rather stringent criteria can be met if the UBP does not cause major perturbations of the major and minor groove interactions of the base pair under construction and the polymerase.…”