Detection of 3′-End RNA Uridylation with a Protein Nanopore

Abstract

Post-transcriptional modifications of the 3′-ends of RNA molecules have a profound impact on their stability and processing in the cell. Uridylation, the addition of uridines to 3′-ends, has recently been found to be an important regulatory signal to stabilize the tagged molecules or to direct them towards degradation. Simple and cost-effective methods for the detection of this post-transcriptional modification are not yet available. Here, we demonstrate the selective and transient binding of 3′-uridylated ssRNAs inside the β barrel of the staphylococcal alpha-hemolysin (αHL) nanopore, and investigate the molecular basis of uridine recognition by the pore. We show the discrimination of 3′-oligouridine tails on the basis of their lengths and propose the αHL nanopore as a useful sensor for this biologically relevant RNA modification.

The evolution of nanopore sequencing

“…This group has proved a mean accuracy of 99.8% for four dNMPs and the ability of 5-mdC identification by engineering α-HL nanopore to facilitate longer dwelling time for accurate detection (Astier et al, 2006 ; Clarke et al, 2009 ). Recently, this group also demonstrated the possibilities of sequencing RNA and detecting RNA uridylation at their 3′ ends by α-HL nanopore (Ayub and Bayley, 2012 ; Ayub et al, 2013 ; Clamer et al, 2013 ; Cracknell et al, 2013 ). The RNA-sequencing expands potential application of nanopore into direct sequencing of transcriptome and detecting of post-transcriptional RNA modification at the single-molecule level.…”

Highly parallel direct RNA sequencing on an array of nanopores

“…Oxford Nanopore Technologies have developed a nanopore-based sensing platform which is capable of sequencing DNA directly without the need to perform an enzymatic synthesis reaction. As with DNA oligonucleotides, strands of RNA can be made to pass through a protein nanopore embedded in a hydrophobic membrane when an electrical potential is applied 17 If a motor protein is used to control the speed of translocation of the RNA strand through the nanopore, a signal can be obtained which is governed by the identity of the RNA strand. The signal changes if the primary sequence of the RNA is changed 18 .…”

Engineering of bacterial toxins for research and medicine

“…Then, from experiments using various αHL mutants linked to a such as β-CD and associated with the use of an exonuclease in the reactive medium, a singlemolecule method for sequencing RNA [162,163] and DNA was developed, with the potential to determine modified bases without the need for chemical or fluorescent labeling [164][165][166][167]. However, at present, the speed at which DNA is translocated though the αHL nanopore is too fast for direct strand sequencing.…”