An athermal approach to mRNA enrichment from total RNA using a self-immolative thioester linked nucleic acids (TENA) is described. Oligo(thymine) (oT) TENA has a six-atom spacing between bases which allowed TENA to selectively base-pair with polyadenine RNA. As a result of the neutral backbone of TENA and the hydrophobicity of the octanethiol end group, oT TENA is water insoluble and efficiently pulled down 93±2 % of EGFP mRNA at a concentration of 10 ng μL-1 . Self-immolative degradation of TENA upon ambient temperature exposure to nucleophilic buffer components (Tris, DTT) allowed recovery of 55±27 ng of mRNA from 3.1 μg of total RNA, which was not statistically different from the amount recovered using Dynabeads® mRNA DIRECT Kit (89±24 ng). Gene expression as measured by RT-qPCR was comparable for both enrichment methods, suggesting that the mild conditions required for enrichment of mRNA using oT TENA are compatible with RT-qPCR and other downstream molecular biology applications.
Keywords: mRNA; oligonucleotides; polymerase chain reaction; polymerization.
© 2021 Wiley-VCH GmbH.