Efficient Reverse Transcription Using Locked Nucleic Acid Nucleotides towards the Evolution of Nuclease Resistant RNA Aptamers

Abstract

BACKGROUND: Modified nucleotides are increasingly being utilized in the de novo selection of aptamers for enhancing their drug-like character and abolishing the need for time consuming trial-and-error based post-selection modifications. Locked nucleic acid (LNA) is one of the most prominent and successful nucleic acid analogues because of its remarkable properties, and widely explored as building blocks in therapeutic oligonucleotides. Evolution of LNA-modified RNA aptamers requires an efficient reverse transcription method for PCR enrichment of the selected RNA aptamer candidates. Establishing this key step is a pre-requisite for performing LNA-modified RNA aptamer selection. METHODOLOGY: In this study three different reverse transcriptases were investigated towards the enzymatic recognition of LNA nucleotides. Both incorporation as well as reading capabilities of the LNA nucleotides was investigated to fully understand the limitations of the enzymatic recognition. CONCLUSIONS: We found that SuperScript® III Reverse Transcriptase is an efficient enzyme for the recognition of LNA nucleotides, making it a prime candidate to be used in de novo selection of LNA containing RNA aptamers.