efficient reverse transcription using locked nucleic acid nucleotides towards the evolution of nuclease resistant rna aptamers有效使用锁核酸逆转录核苷酸抗核酸酶rna寡核苷酸适配子的进化.pdfVIP

Efficient Reverse Transcription Using Locked Nucleic Acid Nucleotides towards the Evolution of Nuclease Resistant RNA Aptamers 1,2 1,2 1 1,3 4 Lucile Crouzier , Camille Dubois , Stacey L. Edwards , Lasse H. Lauridsen , Jesper Wengel , Rakesh N. Veedu1* 1 School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Brisbane, Queensland, Australia, 2 Institute Polytechnique LaSalle Beauvais, Beauvais, France, 3 The Novo Nordisk Foundation Center for Biosustainability, Scion DTU, Hørsholm, Denmark, 4 Nucleic Acid Center, Department of Physics and Chemistry, University of Southern Denmark, Odense, Denmark 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 SuperScriptH III Re