Indirect analysis of oligonucleotides using cleavable small molecule mass tags with detection by mass spectrometry

• Main Author: Riley, Jo-Anne
• Format: Dissertation
• Language: English
• Published: University of Southampton 2013
• Subjects: QD Chemistry

In the 1990s, siRNAs and microRNAs were discovered to be naturally occurring genetic regulators. This provided a new potential mechanism of action for drugs with applicability to a wide range of therapeutic areas. Consequently, a substantial increase into oligonucleotide research has occurred, leading to the need for improved and novel techniques for their analysis. Standard methods of oligonucleotide analysis are based on hybridisation assays with analysis via detection probes labelled with fluorescent tags. However, multiplexing potential is limited due to the broad, and thus often overlapping, signals emitted. An alternative to labelling detection probes with fluorescent tags is to use cleavable small molecule mass tags with detection by mass spectrometry. Herein, a self-reporting detection probe was designed for use in a hybridisation assay for indirect oligonucleotide detection via cleavable small molecule mass tags. The self-reporting detection probe contains an analyte complementary region and a reporter region. The reporter region is a custom designed DNA/RNA chimeric nucleotide sequence. The ribose-phosphate backbone is used as a built-in enzyme cleavable linker, generating small nucleotide products upon cleavage by RNase A. These nucleotides can then serve as mass tags for indirect detection of oligonucleotides. This system avoids the need to design or synthesise a cleavable linker by exploiting the properties of the RNA molecule. This approach was used for the successful detection of a synthetic microRNA and the multiplexing potential was demonstrated by the simultaneous detection of two RNAs.