A novel, dynamic chemistry-based method, developed by Professor Mark Bradley and group, has provided a breakthrough solution for the direct detection of microRNAs - key biomarkers of multiple disease states.
The challenge: unleashing the potential of miRNA biomarkers
MicroRNAs (miRNAs) are powerful biomarkers whose levels in biological fluids are indicative of multiple disease states including cancer, liver failure, and heart disease. The development of a rapid and accurate method for their detection and quantification will provide a crucial tool in clinical trials and drug development and deliver improved diagnosis and treatment of disease. However, miRNAs have been exceptionally problematic to analyse due to their small size, poor stability and the low levels found in plasma – preventing their use as valuable biomarkers.
Dynamic covalent chemistry for error-free analysis of nucleic acids
Research in the Bradley group between 2007 and 2012 led to the development and validation of a novel method of DNA analysis using dynamic covalent chemistry – i.e. reversible bond-formation under thermodynamic control – to enable error-free genetic analysis. Key to the method is the generation of so-called peptide nucleic acid (PNA) oligomers (“probes”) designed to hybridise the target nucleic acid, with an empty position where one of the bases (in the middle of the strand) is deliberately omitted. When the PNA probe hybridises with the target nucleic acid, this empty position creates a “pocket” containing the unknown nucleotide under interrogation (Figure 1). Subsequently, four bespoke synthetic aldehyde-containing nucleobases are added, which go in and out of the pocket, via reversible, dynamic, imine chemistry, to generate a duplex structure with the “best fitting” nucleobase incorporated. These aldehyde-containing nucleobases can be functionalised with fluorophores or biotin to permit the easy detection of their incorporation.
The method guarantees specificity with signal detection only achievable IF the target nucleic acid sequence binds in perfect alignment on the PNA probe, AND the correct aldehyde-base fits into the chemical pocket created by the empty position. It is unique in allowing direct detection and quantification of miRNAs from body fluids (e.g. serum), without the need for PCR amplification processes, which introduce errors and increase time and costs.
The Bradley method for error-free genetic analysis nucleic acid analysis. Image credit: DESTINA.
First commercial direct detection of miRNA via DESTINA Genomics
DESTINA spun out of the University of Edinburgh in 2011 with a worldwide, exclusive license to develop the Bradley group technology. Their resulting ChemiRNA™ Tech is the only method currently available commercially to analyse miRNA directly from patient plasma samples. Studies have shown that it offers a 1,000-fold improvement in miRNA detection limits compared to current methods3.
Investment of €1.2M has been raised to support the commercialisation of the technology and the company rolled out its first assay kit, LiverAce, in 2019. LiverAce is the first assay in the world to offer reliable screening for liver injury based on miRNA and, in clinical studies, has been shown to accurately identify patients at risk of drug induced liver injury after paracetamol overdose.
In addition to LiverAce, DESTINA’s pipeline of assays under development targets lung cancer screening, acute kidney failure, and heart failure. In 2020, a second spin-out company, Vetsina, was created to apply the DESTINA technology for applications in animal health.
- F.R. Bowler, J.J. Diaz-Mochon, M.D. Swift and M. Bradley, “DNA Analysis by Dynamic Chemistry”, Angew. Chem., Int. Ed., 2010, 49, 1809-1812. DOI: 10.1002/anie.200905699.
- F.R. Bowler, P.A. Reid, A.C. Boyd, J.J. Diaz-Mochon and M. Bradley, “Dynamic chemistry for enzyme-free allele discrimination in genotyping by MALDI-TOF mass spectrometry”, Anal. Methods, 2011, 3, 1656-1663. DOI: 10.1039/C1AY05176H.
- B. López-Longarela et al., “Direct Detection of miR-122 in Hepatotoxicity Using Dynamic Chemical Labeling Overcomes Stability and isomiR Challenges”, Anal.l Chem., 2020, 92, 4, 3388–3395. DOI: 10.1021/acs.analchem.9b05449
- M. Bradley and J.J. Diaz-Mochon, “Nucleobase characterisation”, Patent WO 2009/037473, 2009.