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

Area of Science:

• Biotechnology
• Nanotechnology
• Structural Biology

Background:

• RNA origami allows for self-assembling RNA nanostructures for applications in biosensing, synthetic biology, and nanomedicine.
• A key challenge is assessing the structural integrity and folding dynamics of these RNA scaffolds in solution.
• Existing methods for structural readout can be limited in scalability and require specific labeling.

Purpose of the Study:

• To adapt microfluidic modulation spectroscopy (MMS) as a label-free technique for analyzing RNA folding and structure.
• To establish a workflow for quantifying RNA base-pairing states and monitoring structural maturation.
• To demonstrate the utility of MMS for studying RNA origami nanostructures under native conditions.

Main Methods:

• Microfluidic Modulation Spectroscopy (MMS) was employed, utilizing the vibrational fingerprints of RNA bases and base pairs (1760-1600 cm⁻¹).
• A microfluidic transmission cell was used for automated background subtraction, enabling measurements from small sample volumes.
• An analysis workflow involving baselined second derivative and constrained deconvolution was developed to quantify paired and unpaired RNA populations.

Main Results:

• MMS successfully measured RNA folding and structural states in microliter volumes under native solution conditions.
• Thermal ramping experiments revealed distinct unfolding barcodes for different RNA conformational states, distinguishing young and mature ensembles.
• The study tracked the post-transcriptional maturation of RNA origami from a kinetically trapped state to a more compact, base-paired structure.

Conclusions:

• MMS serves as a rapid, automated, and scalable method for structural analysis of RNA origami.
• This technique complements high-resolution methods like cryo-EM and SAXS for engineering dynamic RNA nanostructures.
• MMS facilitates the characterization of RNA folding dynamics, crucial for the development of advanced RNA-based biosensors and nanomedicines.