With a long-term aim of developing a versatile sensing platform for <i>in vitro</i> detection of micro-RNA, we have designed a sensor scaffold based on controlled unfolding of an intramolecular guanine quadruplex through base-pairing mediated target recognition. This quadruplex molecular beacon (QMB) is based on the human telomeric quadruplex and features an elongated central loop that serves as a recognition domain. Binding of a target sequence to the loop triggers unfolding of the quadruplex and structurally separates a FRET pair, leading to a fluorescent signal. To develop a platform that is truly versatile, a greater understanding of how changes in sequence effect both the folded structure and stability of the quadruplex and stimuli-responsive unfolding is needed. In this work, we examined the impacts of FRET pair positioning and loop length on both the quadruplex stability and the responsiveness of the QMB sensor. Quadruplex stability was analyzed through UV-Visible thermal denaturation, with our findings suggesting that internal attachment (via modified thymine residues) substantially increases thermal stability relative to terminal attachment (via phosphate esters). Analysis of sensor responsiveness (relative increase in fluorescence on target recognition) suggests that there is an optimum intermediate loop length (15-16 nt) with both longer and shorter recognition loops yielding lower relative signal intensity. Additionally, these sensors appear to be robust, functioning in both potassium and sodium rich environments as well as in the presence of serum proteins, suggesting they will be amenable to use <i>in vitro</i>.

Impacts of loop length and FRET pair position on stability and function of quadruplex molecular beacons

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