Versatile Enzymatic Approach for Truncation, Labeling, and Mutation of Nucleobase-Modified Aptamers Demonstrated on Indole-Modified β-Conglutin Aptamer

Identificador:  imarina:9466789

Handle: https://hdl.handle.net/20.500.11797/imarina9466789

Autores:  Ondrus, M; Franco-Urquijo, PA; Lerga, TM; Cechová, LM; Redondo, MCB; O'Sullivan, CK; Hocek, M

Resumen:
A new enzymatic method for truncation of aptamers from 5'-end has been developed and demonstrated on a newly selected indole-modified beta-conglutin aptamer. This method relies on extension of a primer containing ribonucleotides, which can be specifically hydrolyzed by RNase, thereby removing the whole or 5'-part of the primer. This approach enables flexible synthesis of modified aptamers truncated from the 5' end, 3' end, or both ends-a capability previously achievable only through chemical oligonucleotide synthesis. Furthermore, by employing doubly labeled primers, diverse labeled or modified nucleotides are introduced to prepare 5'-labeled aptamers suitable for fluorescence- or immobilization-based assays. The different variations of this method also enable the synthesis of mutated sequences to study the effect of modifications and their positions. Overall, this enzymatic method provides a valuable alternative for the truncation or labeling of (hyper)modified and functional oligonucleotides avoiding the need for chemical synthesis.