[Advance in the application of molecular ruler-based RNA 3D dynamic analysis for the interpretation of variants of...

Functional interpretation of genetic variants is a core issue in precision medicine. A large number of "variants of uncertain significance" (VUS) have remained difficult to classify due to a lack of direct evidence showing alterations in protein-coding sequences. Meanwhile, they may cause disease by reshaping the conformational ensembles and energy landscapes of RNA molecules, a mechanism that extends beyond the detection dimension of linear transcriptomics. This article has systematically reviewed unnatural base pair (UBP)-mediated molecular ruler technologies. By employing site-specific labeling in combination with techniques such as pulsed electron double resonance (PELDOR/DEER), single-molecule fluorescence resonance energy transfer (smFRET), and X-ray scattering interferometry (XSI), this approach has enabled the precise measurement of RNA conformational ensembles and dynamic changes across Ångstrom-to-nanometer spatial scales and nanosecond-to-second temporal scales. This article also demonstrates how genetic variants may induce functional dysregulation through physical mechanisms, such as altering RNA energy landscapes, inducing kinetic traps, or affecting translation rates. It further discussed the translational challenges of converting structural dynamic data into PS3/BS3 level functional evidence under the ACMG/AMP guidelines. Finally, a tiered screening strategy that integrates artificial intelligence prediction, high-throughput in-cell chemical probing, Bayesian statistical evaluation, and molecular ruler validation was proposed. This structural dynamics paradigm holds promise of providing an objective physical basis for the precise interpretation of VUS, which may serve as novel functional evidence to support clinical pathogenicity grading, which may offer important insights for the development of RNA-targeted therapeutics.