Enzymatic Synthesis of 2-Amino-adenosine Triphosphate as a Noncanonical Nucleotide Precursor of Z-Modified RNA

2-amino-deoxyadenosine (dZ) occurs naturally in certain bacteriophage genomes, where it replaces deoxyadenosine, forming three hydrogen bonds with thymidine. This noncanonical deoxyribonucleoside underlies the unique biophysical properties of dZ-DNA. Its corresponding ribonucleoside has been introduced to RNA to form Z-modified RNA, with promising applications in vaccine production and biomedicine. Unlike dZ-DNA, Z-modified RNA has only been synthesized in vitro, which required the addition of chemically synthesized 2-amino-adenosine triphosphate (ZTP) as a precursor. Here, we describe enzyme engineering studies on dZMP–succinate-synthetase (PurZ), a key enzyme in the bacteriophage dZ-DNA biosynthetic pathway that natively catalyzes the conversion of deoxyguanosine monophosphate (dGMP) to dZMP–succinate. Through site-saturation mutagenesis, we generated mutants with altered substrate specificity, capable of catalyzing the conversion of GMP to ZMP–succinate. We further demonstrated that these mutants, in combination with bacterial adenylosuccinate lyase, guanylate kinase and nucleoside diphosphate kinase, efficiently convert GMP to ZTP, marking a critical step in developing a biosynthetic pathway for Z-modified RNA, and enabling enzymatic synthesis of ZTP on a semipreparative scale. Our work provides the basis for further research on the impacts of Z-modified RNA in living organisms, and supports the cost-effective production of Z-modified RNA vaccines and therapeutics.