• Fundamentals of phosphorothioate nucleic acids reviewed from synthesis to metal binding. • Applications of phosphorothioate nucleic acids in developing biosensors and chemical biology reviewed. • Applications of phosphorothioate DNA in assembly and the directed growth of nanomaterials reviewed. Phosphorothioate (PS) modification replaces one of the non-bridging oxygen atoms by sulfur in the phosphate backbone of nucleic acids. While PS DNAs have been traditionally used as nuclease-resistant antisense agents and PS RNA as probe of metal binding in ribozymes, multiple new applications have emerged in recent years. In this review, we start by briefly introducing the structure and synthesis of PS nucleic acids followed by their fundamental chemical and biochemical properties. Further, their recently emerged surface science applications are discussed, such as attachment of DNA to various surfaces and nanomaterials containing thiophilic metals such as gold, silver and cadmium, and templating the growth of these materials. Their role in conferring structural effects in the presence of certain metal ions and in fishing out novel aptamers are also discussed. Covalent chemistry can be performed on the sulfur atom for further grafting functional groups to the backbone of DNA. For PS RNA, we discuss their role as probes for metal binding in ribozymes and DNAzymes, which leads to applications in detection of thiophilic metal ions. Since each PS modification site produces a chiral phosphorus center, the synthesis and purification of diastereomers and their applications are emphasized throughout this review. In the end, a few future research directions are discussed.

Ag10c is a recently reported RNA-cleaving DNAzyme obtained from in vitro selection. Its cleavage activity selectively requires Ag+ ions, and thus it has been used as a sensor for Ag+ detection. However, the previous selection yielded very limited information regarding its sequence requirement, since only ∼0.1% of the population in the final library were related to Ag10c and most other sequences were inactive. In this work, we performed a reselection by randomizing the 19 important nucleotides in Ag10c in such a way that a purine has an equal chance of being A or G, whereas a pyrimidine has an equal chance of being T or C. The round 3 library of the reselection was carefully analyzed and a statistic understanding of the relative importance of each nucleotide was obtained. At the same time, a more active mutant was identified, containing two mutated nucleotides. Further analysis indicated new base pairs leading to an enzyme with smaller catalytic loops but with ∼200% activity of the original Ag10c, and also excellent selectivity for Ag+. Therefore, a more active mutant of Ag10c was obtained and further truncations were successfully performed, which might be better candidates for developing new biosensors for silver. A deeper biochemical understanding was also obtained using this reselection method.