Messenger RNA enrichment using synthetic oligo(T) click nucleic acids

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.

Figure 1 –

(a) Comparison of a natural DNA repeat unit to the CNA repeat unit. The thioether backbone removes backbone charge, but the 6-atom spacing allows for binding of complementary nucleic acids. (b) General process of mRNA isolation procedure. The oligo(T) CNA binds to and helps precipitate mRNA in solution and the mRNA can be released through a heating and reconstitution step.

Figure 2 –

Pulldown of A₂₀ RNA as a function of oligo(T) CNA concentration. Oligo(T) CNA at sufficiently high concentrations achieved >90% pulldown of complementary RNA while effectively no pulldown was observed regardless of concentration for non-complementary sequences. (b) Release of RNA is achieved by heating samples to 75°C to dissociate the hybridization between CNA and RNA. Data is represented as the mean of at least 3 replicates and error bars represent standard deviations.

Figure 3 –

(a) Effective pulldown of fluorescent EGFP mRNA was achieved at CNA concentrations of 125 μM and higher. (b) Under optimized conditions, greater than 90% pulldown efficiency could be achieved at biologically relevant mRNA concentrations. (c) In vitro translation of enriched mRNA reveals that complementary CNA is needed for isolation to occur. (d) Enrichment of mRNA is only possible after the heating and reconstitution step. Data is represented as the mean of at least 3 replicates and error bars represent standard deviations.

Figure 4 –

(a) Using optimized buffer conditions, the performance of oligo(T) CNA compared to Dynabeads showed no statistical difference in mass yield. (b) There was also no statistical difference in relative expression levels measured using mRNA input from different isolation procedures. Data is represented as the mean of at least 3 replicates and error bars represent standard deviations.