Molecular Approaches To Address the Challenges of RNA Analysis in Complex Matrices

We present on a design change and addition of an internal polyethylene glycol (PEG) spacer to an existing biosensor. There were two reasons for changing the sensor design. The first was to increase the stability of the biosensor to avoid binding off-analytes with single nucleotide polymorphisms. The second was to prevent sensor degradation by nucleases. The biosensor, designed for detection of short noncoding RNA strands, is composed of Reporter and Probe nucleic acid strands that form a partially complementary duplex. The internal PEG was added to the Reporter, and subsequently diminished false negatives that resulted from off-oligonucleotide binding. Furthermore, the PEG eliminated degradation of the sensor by DNase1 endonuclease. Currently, in situ and crude cell lysate RNA analysis is hindered by nonspecific interactions and degradation by endogenous nucleases. Together, the design changes presented here mitigate these matrix effects and allow for robust RNA analysis in complex media.