SecAntiGami: A Secondary-Antibody-Inspired RNA Origami for Qualitative Aptamer Validation

Abstract RNA aptamers are short, single-stranded RNA molecules that fold into specific three-dimensional structures and selectively bind molecular targets. Despite applications in therapeutics, diagnostics, and biosensing, tools for aptamer validation remain scarce compared to well-established pipelines for other biomolecular binders such as antibodies. Here, we introduce SecAntiGami─a secondary-antibody-inspired RNA origami─as a modular tool for signal enhancement and aptamer characterization. SecAntiGami binds aptamers of interest through programmable kissing-loop interactions and carries a fluorescent aptamer for visualization. It amplifies aptamer-dependent size increases in dynamic light scattering assays and enables fluorescence-based aptamer localization. We rationally design three different variants of a biotin-binding aptamer and use SecAntiGami to demonstrate that all of them retain their functionality, enabling efficient integration of aptamers into RNA origami structures. SecAntiGami serves as a versatile tool for qualitative aptamer validation for synthetic biology, structural RNA nanotechnology, RNA therapeutics, and diagnostics.