Label-Free Microfluidic Modulation Spectroscopy Monitors RNA Origami Structure and Stability.

RNA origami enables genetically encoded, single-stranded RNA nanostructures that can self-assemble through co-transcriptional folding and are increasingly deployed as scaffolds for biosensing, synthetic biology, and nanomedicine. A recurring practical bottleneck is scalable, solution-phase readout of whether a designed scaffold has reached its intended base-paired architecture, whether it undergoes slow maturation or kinetic trapping, and how its stability is distributed across motifs. Here, we adapt microfluidic modulation spectroscopy (MMS) as a label-free structural biosensor for RNA folding by exploiting the rich 1760-1600 cm-1 vibrational fingerprints of RNA bases and base pairs. MMS alternates between sample and composition-matched buffer measurements in a microfluidic transmission cell to automatically subtract the solvent background, enabling high-quality spectral measurement from microliter volumes under native solution conditions. Using a six-helix-bundle-with-clasp (6HBC) RNA origami as a model, we established an analysis workflow (baselined second derivative and constrained deconvolution) to quantify paired versus unpaired populations. Thermal ramping resolves multiple unfolding events and yields an unfolding barcode that differs between young and mature ensembles. Importantly, MMS tracks post-transcriptional maturation from a kinetically trapped young conformer toward a more compact, base-paired mature state, consistent with prior cryo-EM/SAXS observations for 6HBC RNA origami. Together, these results position MMS as a rapid, automated, and scalable complement to high-resolution structure determination for engineering dynamic RNA origami biosensors.