Observed versus predicted structure of fluorescent self-quenching reporter molecules (SQRM): Caveats with respect to the use of “stem–loop” oligonucleotides as probes for mRNA folding

We developed self-quenching reporter molecules (SQRMs), oligodeoxynucleotides with fluorophore and quencher moieties at the 5′ and 3′ ends respectively, to probe mRNAs for single-stranded, hybridization accessible sequences. SQRMs and their homologous antecedents, Molecular Beacons (MB), are designed with the assumption that they adopt a stem–loop structure thought critical for regulating their reporter function. Recently, we observed that stem–loop structures are not required for SQRM function, and on this basis proposed that these reporter molecules be classified according to whether they were stemmed (Type I) or not (Type II). This finding further stimulated us to investigate whether Type I SQRMs, and by extension MBs, actually adopt a stem–loop configuration under physiologic conditions. Accordingly, we synthesized Type I and Type II SQRMs and studied the thermodynamic characteristics of each by fluorescence melting analysis. The results of these studies suggested that the majority of stem–loop Type I SQRMs are unstructured at 37°C, while some of the stemless Type II SQRMs are, surprisingly, structured. These results were not predicted by the mfold computer program. Type I and II SQRMs were then employed to “map” the mRNA secondary structure of a gene encoding a tyrosine kinase receptor, c-kit. Neither experimentally determined melting temperatures nor mfold-“predicted” thermodynamic parameters were useful in predicting the fluorescence signal-to-noise ratios obtained for SQRMs incubated with c-kit mRNA. We conclude that stem–loop reporter molecules are in fact unlikely to adopt their presumed structures at 37°C, and this design consideration may be dispensed when their use is contemplated under physiologic conditions.