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.
Highlights
Introduction to Unnatural Base PairsGenetic information in all living organisms is encoded in DNA, which consists of nucleotides with four different nucleobases that form nucleobase pairs
The dP–dZ pair is based on an alternative hydrogen-bonding pattern compared with the natural base pairs dA–dT and dG–dC and adopts similar structures in free duplex DNA and the active site of KlenTaq DNA polymerase as the natural pairs
The fidelity of replication by DNA polymerases is still lower for dP–dZ than for the discussed hydrophobic unnatural base pairs (UBPs), mainly owing to the higher propensity for mispairing with natural nucleotides
Summary
A 99.9 % selectivity in turn leads to 97 % retention of the UBP after 30 cycles of PCR (0.99930 = 0.97) and only 90 % retention after 100 cycles of PCR Even though this degree of selectivity is sufficient for a number of applications (e.g., in the use of primers containing UBPs in nested PCR or use in diagnostics),[13,14] for others, where high amplification of the DNA or plasmid containing the UBP is performed and loss of the UBP is critical (e.g., if implemented in an SSO that should produce proteins containing an unnatural amino acid),[9] a selectivity truly approaching that of natural pairs is crucial. DNA templates containing up to four consecutive dP–dZ pairs can be PCR amplified by Taq and Phusion DNA polymerases.[30]
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