Self-reporting hybridisation assay for miRNA analysis.

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.

Self reporting RNA probes as an alternative to cleavable small molecule mass tags.

The large size of biological molecules such as proteins and oligonucleotides makes them inherently problematic to analyse and quantify directly by mass spectrometry. For these molecules, electrospray ionisation produces multiply charged species and associated alkali metal adducts which can reduce sensitivity and complicate quantification. Whereas time-of-flight mass analysers, often coupled to matrix-assisted laser desorption/ionisation, can have insufficient mass resolution to resolve these large molecules in the higher m/z range. This has led to the development of cleavable small molecule mass tag approaches for the indirect analysis of biomolecules such as proteins and oligonucleotides. Existing methodologies require the design and synthesis of a cleavable linker to join the biomolecule and the mass tag. Here, an alternative approach to small molecule mass tags is presented, which exploits the properties of the RNA molecule to afford self-reporting probes which can be easily synthesised using automated phosphoramidite chemistry. The sugar-phosphate backbone of RNA was used as a built-in enzyme cleavable linker and through the use of RNase digestion of bromine labelled oligonucleotides the observation of a range of small molecule mass tags by mass spectrometry is demonstrated. This study provides a proof-of-concept that RNase digestion can be used to produce labelled small molecule mass tags from oligonucleotide probes, thus eliminating the need for custom design and synthesis of a cleavable linker.

Can density functional theory (DFT) be used as an aid to a deeper understanding of tandem mass spectrometric fragmentation pathways?

Prediction of tandem mass spectrometric (MS/MS) fragmentation for non-peptidic molecules based on structure is of immense interest to the mass spectrometrist. If a reliable approach to MS/MS prediction could be achieved its impact within the pharmaceutical industry could be immense. Many publications have stressed that the fragmentation of a molecular ion or protonated molecule is a complex process that depends on many parameters, making prediction difficult. Commercial prediction software relies on a collection of general heuristic rules of fragmentation, which involve cleaving every bond in the structure to produce a list of 'expected' masses which can be compared with the experimental data. These approaches do not take into account the thermodynamic or molecular orbital effects that impact on the molecule at the point of protonation which could influence the potential sites of bond cleavage based on the structural motif. A series of compounds have been studied by examining the experimentally derived high-resolution MS/MS data and comparing it with the in silico modelling of the neutral and protonated structures. The effect that protonation at specific sites can have on the bond lengths has also been determined. We have calculated the thermodynamically most stable protonated species and have observed how that information can help predict the cleavage site for that ion. The data have shown that this use of in silico techniques could be a possible way to predict MS/MS spectra.