Rational design of terminal deoxynucleotidyl transferase for RNA primer elongation

The short synthetic oligonucleotides have laid foundations for modern digital biology, biomaterial, and new therapeutics. However, our abilities to synthesize arbitrary sequences of oligonucleotides were stifled by the decades old phosphoramidite chemistry. The template-independent polymerase, Terminal Deoxynucleotidyl Transferase (TdT), is central to de novo enzymatic DNA synthesis through extensive engineering at the substrate binding site, yet the engineered TdT remained inaccessible to the majority of RNA primers. Here we rationally engineered the primer recognition site of TdT for RNA-primed polymerization. We demonstrated the elevation of RNA elongation activity from 20 % to >90 % on a diverse set of primers and evaluated the reaction dynamics. Pairing with the natural nucleotide substrates, the designed R-TdTs could simplify the analytical procedure for RNA sequences. We developed two proof-of-principle methods for feasible detection of trace amount of microRNAs. Combined with the versatile mutations at substrate binding pocket to accommodate nucleotide building blocks, our designed RNA-editing enzymes would become easily adaptable for a wide range of future applications in de novo synthesis of nucleic acid and synthetic biology.