Phosphorothioate (PS) modification refers to replacing one of the nonbridging oxygen atoms in nucleic acids with sulfur. PS modifications can be easily introduced during solid-phase DNA synthesis. It has been extensively used in ribozyme and DNAzyme research to achieve a bioinorganic understanding of metal binding, bioanalytical applications of metal detection, and chemical biology of DNA modification. It allows for the access of new chemistry, not available to natural DNA. Since each PS modification is accompanied by the production of a chiral phosphorus center, a key technical challenge is to separate the two diastereomers called Rp and Sp. In this chapter, we describe our methods of HPLC-based separation followed by ligation to generate a long and fluorescently modified DNAzyme substrate. Subsequently, the use of the modified substrate for activity assay to understand metal binding and for metal ion detection is also described.
Keywords: Biosensors; DNAzymes; Deoxyribozymes; Phosphorothioate; RNA.
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