Detection of prolactin inducible protein mRNA, a biomarker for breast cancer metastasis, using a molecular beacon-based assay.

Mortality due to breast cancer is increasingly linked to early, undetected metastasis, making methods for earlier detection acutely necessary. We describe the development of an assay based on molecular beacon (MB) chemistry with fluorescence detection to monitor a breast cancer biomarker for the analysis of breast cancer metastasis. The MB assay is based on the complementary base-pairing interactions of the MB nucleic acid with mRNA indicative of breast cancer metastasis. The presence of mRNA is characterized by an increase in the fluorescence intensity of the molecular beacon. The assay gives a linear, reproducible response to prolactin inducible protein mRNA, with a limit of detection in the high picomolar range. This method sensitively and specifically identifies a biomarker directly in serum samples in minimal time and with a straightforward procedure, dramatically reducing the total time for sample analysis over current methods from days to hours. The potential impact of this work in detection and understanding of breast cancer metastasis lies in improvements in simplicity, accuracy, and speed over current methods, which could allow for improved patient treatment and prognoses. Ultimately, additional sample throughput will result in better understanding of disease progression.

Assay for glucosamine 6-phosphate using a ligand-activated ribozyme with fluorescence resonance energy transfer or CE-laser-induced fluorescence detection.

A naturally occurring aptazyme, the glmS ribozyme, is adapted to an assay for glucosamine 6-phosphate, an effector molecule for the aptazyme. In the assay, binding of analyte allosterically activates aptazyme to cleave a fluorescently labeled oligonucleotide substrate. The extent of reaction, and hence analyte concentration, is detected by either fluorescence resonance energy transfer (FRET) or capillary electrophoresis with laser-induced fluorescence (CE-LIF). With FRET, assay signal is the rate of increase in FRET in presence of analyte. With CE-LIF, the assay signal is the peak height of cleavage product formed after a fixed incubation time. The assay has a linear response up to 100 (CE-LIF) or 500 microM (FRET) and detection limit of approximately 500 nM for glucosamine 6-phosphate under single-turnover conditions. When substrate is present in excess of the aptazyme, it is possible to amplify the signal by multiple turnovers to achieve a 13-fold improvement in sensitivity and detection limit of 50 nM. Successful signal amplification requires a temperature cycle to alternately dissociate cleaved substrate and allow fresh substrate to bind aptazyme. The results show that aptazymes have potential utility as analytical reagents for quantification of effector molecules.

Trans-acting glmS catalytic riboswitch: locked and loaded.

A recently discovered class of gene regulatory RNAs, coined riboswitches, are commonly found in noncoding segments of bacterial and some eukaryotic mRNAs. Gene up- or down-regulation is triggered by binding of a small organic metabolite, which typically induces an RNA conformational change. Unique among these noncoding RNAs is the glmS catalytic riboswitch, or ribozyme, found in the 5'-untranslated region of the glmS gene in Gram-positive bacteria. It is activated by glucosamine-6-phosphate (GlcN6P), leading to site-specific backbone cleavage of the mRNA and subsequent repression of the glmS gene, responsible for cellular GlcN6P production. Recent biochemical and structural evidence suggests that the GlcN6P ligand acts as a coenzyme and participates in the cleavage reaction without inducing a conformational change. To better understand the role of GlcN6P in solution structural dynamics and function, we have separated the glmS riboswitch core from Bacillus subtilis into a trans-cleaving ribozyme and an externally cleaved substrate. We find that trans cleavage is rapidly activated by nearly 5000-fold to a rate of 4.4 min(-1) upon addition of 10 mM GlcN6P, comparable to the cis-acting ribozyme. Fluorescence resonance energy transfer suggests that this ribozyme-substrate complex does not undergo a global conformational change upon ligand binding in solution. In addition, footprinting at nucleotide resolution using terbium(III) and RNase V1 indicates no significant changes in secondary and tertiary structure upon ligand binding. These findings suggest that the glmS ribozyme is fully folded in solution prior to binding its activating ligand, supporting recent observations in the crystalline state.