Development of unnatural base pairs (UBPs) has significantly expanded the genetic alphabet both in vitro and in vivo and led to numerous potential applications in the biotechnology and biopharmaceutical industry. Efficient synthesis of oligonucleotides containing unnatural nucleobases is undoubtedly an essential prerequisite for making full use of the UBPs, and de novo synthesis of oligonucleotides with terminal deoxynucleotidyl transferases (TdTs) has emerged as a method of great potential to overcome limitations of traditional solid-phase synthesis. Herein, we report the efficient template-independent incorporation of nucleotides of unnatural nucleobases dTPT3 and dNaM, which have been designed to make one of the most successful UBPs to date, dTPT3-dNaM, into DNA oligonucleotides with a TdT enzyme under optimized conditions. We also demonstrate the efficient TdT incorporation of dTPT3 derivatives with different functional linkers into oligonucleotides for orthogonal labeling of nucleic acids and applications thereof. The development of a method for the daily laboratory preparation of DNAs with UBPs at arbitrary sites with the assistance of TdT is also described.

Backbone-modified nucleic acids are usually more stable enzymatically than their natural counterparts, enabling their broad application as potential diagnostic or therapeutic agents. Moreover, the development of nucleic acids with unnatural backbones has expanded the pool of genetic information carriers and paved the way toward synthetic xenobiology. However, synthesizing these molecules remains very challenging due to the requirement for harsh reaction conditions and the low coupling efficiency during their traditional solidphase synthesis. Although enzymatic synthesis provides an attractive alternative that also allows the replication and artificial evolution of these molecules, it is crucially dependent on the availability of polymerases capable of synthesizing these backbone-modified nucleotides. Previously, a series of thermostable polymerases that can efficiently synthesize or even amplify backbonemodified DNA or RNA have been evolved through a polymerase evolution method based on phage display. Herein we summarize protocols to use these evolved polymerase mutants to transcribe, reverse transcribe, and PCR amplify backbone-modified nucleic acids. We also outline the polymerase chain transcription method, developed later for the rapid production of RNA or backbonemodified RNA with one of these evolved polymerases, SFM4-3.