Self-reporting hybridisation assay for miRNA analysis

Abstract

Hybridisation assays, which are commonly used to analyse oligonucleotides such as siRNAs and miRNAs, often employ detection probes with fluorescent tags. The signal emitted by a fluorescent tag covers a broad range of wavelengths and this limits the multiplexing potential due to overlapping signals. A novel method of indirect oligonucleotide analysis has been developed which combines a hybridisation assay with cleavable small molecule mass tags using HPLC-ESI MS detection. A self-reporting detection probe has been designed which incorporates a DNA/RNA chimeric oligonucleotide sequence in the reporter region, which generates small nucleotide products upon RNase cleavage of the ribose-phosphate backbone. These small nucleotides can then serve as mass tags for the indirect detection of oligonucleotide analytes. The narrow mass range covered by a small molecule mass tag combined with the wide range of possible mass tags provides a high degree of multiplexing potential. This approach has been demonstrated for the analysis of a synthetic miRNA.

A Combination of DNA-peptide Probes and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS): A Quasi-Targeted Proteomics Approach for Multiplexed MicroRNA Quantification

“…On the other hand, traditional spectral separation that often requires a difference in absorbance, fluorescence, refractive index or reflectivity of pre-treated miRNAs could be more difficult. For instance, signals produced by fluorophores are broad, typically covering around 100 nm in a wavelength window of approximately 600 nm 34 . Thus, multiplexing potential of current techniques is highly limited due to overlapping signals.…”

“…Comparatively, signals from mass spectrometry cover in the region of 6 Da, taking into account all isotopic contributions, in a mass window of approximately 1000 Da. This feature has facilitated the simultaneous analysis of many mass tags 34 . In addition, two filtering stages (i.e., precursor ion and product ion) combined with high duty cycle in LC-MS/MS-based targeted proteomics, lead to quantification with unmatched specificity 44 .…”

“…This issue could be magnified intensely if a pool of miRNAs is targeted. Other strategies such as small molecule reporter tags 33 , 34 could have a rapid cost increase with the involvement of more miRNAs due to the chemical property of tags 35 .…”

A Quasi-direct LC-MS/MS-based Targeted Proteomics Approach for miRNA Quantification via a Covalently Immobilized DNA-peptide Probe

“…However, fluorescence signals are easily affected by temperature and pH 24 . In addition, spectral separation often requires a difference in fluorescence for multiplex analysis, which could be difficult 25 . Direct detection techniques are promising, but they are primarily in the early stages of development and still require significant efforts to make them applicable.…”

Quantification of microRNA by DNA–Peptide Probe and Liquid Chromatography–Tandem Mass Spectrometry-Based Quasi-Targeted Proteomics

“…However, the application encountered some difficulties, probably because of complicated and unresolved mass spectra of miRNAs, , especially that miRNAs consist of only four nucleotides that the risk of different sequences giving rise to similar mass spectra patterns is potentially greater than for those molecules containing amino acids . Alternative strategies, including small molecule reporter tags , and high-resolution mass spectrometers, either lack multiplexing capability or are expensive. However, we cannot arbitrarily rule out the use of mass spectrometry in miRNA quantification.…”