Messenger RNA enrichment using synthetic oligo(T) click nucleic acids. Issue 90 (23rd October 2020)

• Record Type: Journal Article
• Authors: Anderson, Alex J.; Culver, Heidi R.; Prieto, Tania R.; Martinez, Payton J.; Sinha, Jasmine; Bryant, Stephanie J.; Bowman, Christopher N.
• Is Part Of: Chemical communications. Volume 56:Issue 90(2020)
• Page Start: 13987
• Page End: 13990
• Publication Date: 2020-10-23
• Subjects: Chemistry -- Periodicals; 540
• DOI: 10.1039/d0cc05815g
• Languages: English
• ISSNs: 1359-7345

Abstract

Abstract : Messenger RNA enrichment is a key step in many molecular biology techniques. Herein, novel poly(T) oligonucleotides, synthesized via cost-effective thiol–ene polymerization, enrich mRNA in yields that rival commercially available techniques Abstract : Enrichment of mRNA is a key step in a number of molecular biology techniques, particularly in the rapidly growing field of transcriptomics. Currently, mRNA is isolated using oligo(thymine) DNA (oligo(dT)) immobilized on solid supports, which binds to the poly(A) tail of mRNA to pull the mRNA out of solution through the use of magnets or centrifugal filters. Here, a simple method to isolate mRNA by complexing it with synthetic click nucleic acids (CNAs) is described. Oligo(T) CNA bound efficiently to mRNA, and because of the insolubility of CNA in water, >90% of mRNA was readily removed from solution using this method. Simple washing, buffer exchange, and heating steps enabled mRNA's enrichment from total RNA, with a yield of 3.1 ± 1.5% of the input total RNA by mass, comparable to the yield from commercially available mRNA enrichment beads. Further, the integrity and activity of mRNA after CNA-facilitated pulldown and release was evaluated through two assays. In vitro translation of EGFP mRNA confirmed the translatability of mRNA into functional protein and RT-qPCR was used to amplify enriched mRNA from total RNA extracts and compare gene expression to results obtained using commercially available products.