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Here, a DNA scaffold\u2010based labeling strategy is developed, and applied to the frameshift\u2010stimulating RNA pseudoknot from the SARS\u2010CoV\u20102 genome. FRET\u2010active reporters were prepared containing both Cy3 (donor) and Cy5 (acceptor) molecules and measurements conducted on freely\u2010diffusing single molecules, enabling the evaluation of conformational heterogeneity via smFRET population distributions. Freely diffusing pseudoknots, modified at the base of stem 1, display a broad range of NaCl\u2010dependent FRET states in solution, consistent with conformational freedom that extends beyond the static X\u2010ray and cryo\u2010EM structures. This work is a proof\u2010of\u2010principle demonstration of the feasibility of our DNA scaffold approach in enabling smFRET studies on this important class of biomolecule. Together, this work outlines new biochemical and biophysical approaches toward the study of RNA conformational dynamics in pseudoknots, riboswitches, and other structured RNA elements.<\/jats:p>","DOI":"10.1002\/cphc.202500568","type":"journal-article","created":{"date-parts":[[2025,11,8]],"date-time":"2025-11-08T08:37:15Z","timestamp":1762591035000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A DNA Scaffold Approach Facilitates 5\u2032 Labeling of the SARS\u2010CoV\u20102 RNA Pseudoknot for Single\u2010Molecule F\u00f6rster Resonance Energy Transfer Investigation"],"prefix":"10.1002","volume":"26","author":[{"given":"Sarah P.","family":"Graham","sequence":"first","affiliation":[{"name":"School of Physics, Engineering and Technology University of York  Heslington YO10 5DD UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jemma 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