Efforts Toward XNA PCR: Optimization and Characterization of PCR-Amplification of Chimeric 2'F XNA/DNA

Xeno Nucleic Acids (XNA) are chemically modified versions of DNA. These modifications can bypass natural immune responses and impart novel properties in complex biological systems. Previous efforts have identified mutant proteins capable of robust XNA synthesis; one mutant, SFM4-3, has been shown to be able to amplify partially substituted XNA in the polymerase chain reaction (PCR) which is necessary for many applications of XNA. However, SFM4-3 mediated PCR is inefficient and has required up to 22 h of reaction time to create amplicons of ∼75 nucleotides (nt); optimizing the PCR amplification of XNA can potentially improve its utility. Here, we show an optimized method for XNA PCR and compare the XNA PCR ability of mutant XNA polymerase enzymes. We show that SFP1 and SFP4 perform better than SFM4-3 in the amplification of 2'F-XNA. We also highlight the role of protein stability in XNA polymerase PCR performance, demonstrating that significantly diminished thermostability may limit current XNA polymerases. This work decreases the time needed for XNA PCR to <4 h, increases the length of amplicons generated to 245 nt, identifies new PCR-active enzymes, and provides guidance for how to improve XNA polymerases for PCR in the future.