Putative G-Quadruplex Structures in Cancer-Dysregulated Circulating lncRNAs and their G4-mediated Identification of Protein Interacting Partners

Circulating long non-coding RNAs (lncRNAs) have emerged as compelling cancer biomarkers. However, the structural features that mediate their extracellular stability and protein interactions remain largely unexplored. Here, we present the first systematic investigation of G-quadruplex (G4) motifs within cancer-dysregulated circulating lncRNAs and exploit these structures as molecular handles to identify associated RNA-binding protein (RBP) networks. From 283 circulating lncRNAs curated from the Lnc2Cancer 3.0 database, putative G-quadruplex-forming sequences (PQSs) were identified computationally using QGRS Mapper and G4Hunter, yielding four prioritized candidates -- AGAP2-AS1, LINC00683, DLG1-AS1, and KRTAP5-AS1 -- spanning 2G to 4G architectures. In vitro transcribed PQSs were validated for parallel G4 formation by circular dichroism spectroscopy, native polyacrylamide gel electrophoresis with thioflavin T staining, and reverse transcriptase stop assays, conducted under both standard buffer and simulated body fluid conditions to approximate the circulatory milieu. Electrophoretic mobility shift assays and isothermal titration calorimetry demonstrated nanomolar-affinity interactions between the G4-containing RNA and human serum albumin (HSA), the most abundant circulating protein. Cross-referencing G4-interacting proteins from the G4IPDB database with lncRNA-protein associations from LncTarD and NPInter, combined with RPISeq interaction predictions, identified ten candidate RBPs. A STRING-based protein-protein interaction (PPI) network was constructed at a confidence threshold of [≥]0.7 and refined iteratively using experimental stability data to exclude proteins associated exclusively with the structurally weaker KRTAP5-AS1. The resulting network, centered on ELAVL1, IGF2BP1, hnRNPA2B1, and FUS, highlights a coordinated post-transcriptional regulatory module relevant to oncogenesis. This work establishes a novel, experimentally validated framework wherein G4 motifs serve as entry points for decoding the protein interactome of circulating lncRNAs, with implications for cancer diagnostics and RNA-targeted therapeutic strategies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/728349v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1bba7dborg.highwire.dtl.DTLVardef@1095a53org.highwire.dtl.DTLVardef@1092eddorg.highwire.dtl.DTLVardef@1e3b7e7_HPS_FORMAT_FIGEXP M_FIG C_FIG