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

Lucile Crouzier,Stacey L Edwards,Rakesh N Veedu,Jesper Wengel,Camille Dubois,Lasse H Lauridsen,Maxim Antopolsky

doi:10.1371/journal.pone.0035990

Abstract

BackgroundModified 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.MethodologyIn 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.ConclusionsWe 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

Highlights

Nucleic acid-based therapeutic approaches have gained significant interests in recent years towards the development of drugs against many diseases
We found that SuperScriptH III Reverse Transcriptase is an efficient enzyme for the recognition of Locked nucleic acid (LNA) nucleotides, making it a prime candidate to be used in de novo selection of LNA containing RNA aptamers
Locked Nucleic Acid (LNA) is one of the most prominent and successful nucleic acid analogues because of its remarkable properties, and it is widely explored as building blocks in therapeutic oligonucleotides [11,12,13,14]

Summary

Introduction

Nucleic acid-based therapeutic approaches have gained significant interests in recent years towards the development of drugs against many diseases. The experiments demonstrated that SuperScriptH III Reverse Transcriptase efficiently incorporated LNA-T nucleotides, encoded by RNA nucleotides of the template strand, and are capable of producing full-length cDNA products in good yield (Figure 2B, lane 4 & C).

Results

Conclusion