Abstract
Transfer RNAs (tRNA) are the most common RNA molecules in cells and have critical roles as both translators of the genetic code and regulators of protein synthesis. As such, numerous methods have focused on studying tRNA abundance and regulation, with the most widely used methods being RNA-seq and microarrays. Though revolutionary to transcriptomics, these assays are limited by an inability to encode tRNA modifications in the requisite cDNA. These modifications are abundant in tRNA and critical to their function. Here, we describe proof-of-concept experiments where individual tRNA molecules are examined as linear strands using a biological nanopore. This method utilizes an enzymatically ligated synthetic DNA adapter to concentrate tRNA at the lipid bilayer of the nanopore device and efficiently denature individual tRNA molecules, as they are pulled through the α-hemolysin (α-HL) nanopore. Additionally, the DNA adapter provides a loading site for ϕ29 DNA polymerase (ϕ29 DNAP), which acts as a brake on the translocating tRNA. This increases the dwell time of adapted tRNA in the nanopore, allowing us to identify the region of the nanopore signal that is produced by the translocating tRNA itself. Using adapter-modified Escherichia coli tRNAfMet and tRNALys, we show that the nanopore signal during controlled translocation is dependent on the identity of the tRNA. This confirms that adapter-modified tRNA can translocate end-to-end through nanopores and provide the foundation for future work in direct sequencing of individual transfer RNA with a nanopore-based device.
Highlights
Transfer RNAs decode genetic information, delivering to the protein-synthesizing ribosome the individual amino acids specified by each codon of a messenger RNA
Capture and Threading of Transfer RNAs (tRNA) Through the α-HL Nanopore is Facilitated by Ligation of an Oligonucleotide Adapter to the tRNA Reading the nucleotide composition of individual tRNA molecules will require capture, denaturation, and threading of each strand sequentially through the nanopore
We found that native tRNA molecules caused long (>30 s) ionic current blockades of the α-HL pore
Summary
Transfer RNAs (tRNA) decode genetic information, delivering to the protein-synthesizing ribosome the individual amino acids specified by each codon of a messenger RNA. It is unsurprising that they are the most numerous RNA species in the cell, composing approximately 80% of the RNA molecules per generation in yeast (Phizicky and Hopper, 2010) These small, non-coding RNA molecules contain numerous post-transcriptionally modified nucleotides, which contribute to tRNA fold stabilization, codon recognition, and aminoacylation [reviewed in Gustilo et al (2008) and Motorin and Helm (2010)]. Current methods for analysis of tRNAs, and RNA in general, include RNA-seq, microarray, and mass spectrometry. These methods are proven tools for detection of novel tRNAs and global tRNA expression patterns (Dittmar et al, 2006; Chan et al, 2011).
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