Quantification of tRNA fragments by electrochemical direct detection in small volume biofluid samples

Hazel Mcardle,Marion C Hogg,David C Henshall,Jochen H M Prehn,Elaine Spain,Kellie Adamson,Sebastian Bauer,Felix Rosenow

doi:10.1038/s41598-020-64485-4

Abstract

Elevated levels of transfer RNA (tRNA) fragments were recently identified in plasma samples from people with epilepsy in advance of a seizure, indicting a potential novel class of circulating biomarker. Current methods for detection and quantitation of tRNA fragments (tRFs) include northern blotting, RNA sequencing or custom Taqman-based PCR assays. The development of a simple, at home or clinic-based test, would benefit from a simple and reliable method to detect the tRFs using small volumes of biofluids. Here we describe an electrochemical direct detection method based on electrocatalytic platinum nanoparticles to detect 3 specific tRFs: 5’AlaTGC, 5'GlyGCC, and 5'GluCTC. Using synthetic tRF mimics we showed this system was linear over 9 orders of magnitude with sub-attomolar limits of detection. Specificity was tested using naturally occurring mismatched tRF mimics. Finally, we quantified tRF levels in patient plasma and showed that our detection system recapitulates results obtained by qPCR. We have designed a tRF detection system with high sensitivity and specificity capable of quantifying tRFs in low volumes of plasma using benchtop apparatus. This is an important step in the development of a point-of-care device for quantifying tRFs in whole blood.

Highlights

Elevated levels of transfer RNA fragments were recently identified in plasma samples from people with epilepsy in advance of a seizure, indicting a potential novel class of circulating biomarker
We recently identified fragments derived from transfer RNAs as novel biomarkers in plasma from people with epilepsy, where we found them to be significantly elevated in advance of a seizure occurring4. tRNAs are one of the most abundant classes of RNAs in the cell, second only to ribosomal RNAs, which function in the same pathway[5]
We recently discovered that specific 5'tRNA fragments (5'tRFs) are elevated in plasma collected from people with epilepsy in advance of seizures and return to baseline levels rapidly post seizure[4]

Summary

Introduction

Elevated levels of transfer RNA (tRNA) fragments were recently identified in plasma samples from people with epilepsy in advance of a seizure, indicting a potential novel class of circulating biomarker. In previous studies we have shown that direct detection of small noncoding RNAs, including miRNA-134, using platinum nanoparticles (PtNPs) resulted in attomolar detection limits in patient plasma samples and showed good correlation with Taqman-based qPCR quantification indicating this technique is suitable for low abundance RNA biomarker quantification[8]. Recent advances to this method led to the development of “TORNADO”, a theranostic one-step RNA detector capable of quantifying miRNA levels in unprocessed patient plasma and cerebrospinal fluid samples[9]. We tested whether three epilepsy-associated 5′tRFs can be detected in biofluids and replicate earlier findings that plasma tRF levels are elevated in advance of seizures

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