In-cell NMR reveals the first direct observation of endogenous interaction between HIV Tat protein and Tat RNA aptamer in human cells

RNA-protein interactions lie at the basis of numerous regulatory and functional cellular biological processes, including transcriptional control, RNA processing, nuclear export, and viral replication. Despite their fundamental biological significance, direct structural investigation of RNA-protein complexes in live human cells remains an unresolved problem due to resolution limits in spatial information, delivery of molecules, and real-time monitoring under native conditions. Existing studies rely on pre-existing in vitro complexes added to cells and therefore overlook important aspects of endogenous binding and localization. Here, we report the first in-cell nuclear magnetic resonance (NMR) study of the de novo formation of an RNA-protein complex in living human cells. By using a model system involving the HIV-1 Tat protein and its high-affinity RNA aptamer, we expressed Tat endogenously in HeLa cells and introduced the aptamer by electroporation. Direct observation was made of native complex formation within the intracellular milieu. In-cell NMR spectra exhibited characteristic chemical shift perturbations and nuclear Overhauser effect (NOE) signatures indicative of specific RNA-protein binding under physiological conditions. Comparison directly with in vitro spectra confirmed structural integrity and binding specificity in the intracellular environment. Remarkably, we obtained a partial NOE-based assignment of the RNA upon complexation with Tat in living cells-an unprecedented step towards cellular structural biology. Complementary confocal microscopy validated nuclear co-localization, enabling functionally relevant interaction. This work shows the first direct, real-time evidence for native RNA-protein complex assembly in human cells. It provides a new paradigm for probing RNA-mediated regulatory events in vivo and expands the horizon of therapeutic RNA design.