A)Folding kinetics of the TERRA G-quadruplex in solutions without ligands (black) or with cPDS+BG4 (red)

A)Folding kinetics of the TERRA G-quadruplex in solutions without ligands (black) or with cPDS+BG4 (red). consist of a stack of planar guanine tetramers called G-quartets that are stabilized by Hoogsteen hydrogen bonds and monovalent cation coordination.[3] G-quadruplex-forming sequences are prevalent in the human genome[46] and are particularly enriched at telomeres and in the promoter regions of genes.[7,8] The formation of stable G-quadruplex structures in the telomeric 3 overhang has been shown to have an inhibitory effect on telomerase, an enzyme up-regulated in a majority of cancer cells.[9] Therefore, the design of small-molecule ligands that can selectively bind and stabilize DNA G-quadruplexes (GQs) in cells has been intensively investigated as a potential strategy for cancer therapy.[10] It has been shown that mammalian telomeres can be transcribed into telomeric repeat-containing RNA (TERRA),[11] which can also form Firocoxib G-quadruplexes in vivo.[12] More generally, RNA G-quadruplexes have been shown to regulate biological processes such as translation.[13,14] This provides a new route to control these biological processes by using molecules that selectively bind to the RNA GQs. We recently exhibited that RNA G-quadruplexes can form in the cytoplasm of cells and that they can be stabilized and visualized by the selective RNA GQ ligand carboxypyridostatin (cPDS)[15] and the antibody BG4.[16] These observations have Firocoxib led to the possibility of a multifaceted regulatory approach, for example, through antibodydrug conjugates (ADCs).[17] It is still unclear whether BG4 and cPDS can cooperatively bind and stabilize an RNA GQ structure in a cellular context. A ternary complex in which an antibody and a small molecule can cooperatively stabilize a GQ would offer a novel approach to target and stabilize these structures and support the observed increase in BG4 staining upon treatment with G-quadruplex ligands. Furthermore, it would be of great significance to see whether the conformational rearrangement widely observed in proteins[1820] and other nucleic acid structures[21,22] are also observed upon binding of ligands to G-quadruplexes. In this report, we investigated the dual binding of cPDS and the BG4 antibody to the TERRA G-quadruplex. Using a mechanical unfolding approach with laser tweezers, we found that a minor TERRA G-quadruplex population (48 %) has increased mechanical and thermodynamic stability when bound to both ligands. With force-jump kinetic investigations, we revealed that the two ligands compete for the binding initially, followed by a slow rearrangement that leads to the formation of the ternary complex. This behavior suggests a conformational transition during binding, which leads to increased stability of the bound TERRA GQ. We anticipate that this new binding strategy may inspire the development of ligands with more effective binding to specific G-quadruplex structures. To carry out single-molecule mechanical unfolding experiments, the GQ-forming sequence 5-UUA(GGG UUA)4-3 (TERRA-G4) was sandwiched BMP6 between two double-stranded DNA/RNA hybrid spacers, which were separately attached to two optically trapped polystyrene beads in a laser tweezers instrument. The entire nucleic acid construct was mechanically stretched and relaxed (Physique1 A) in a 10 mmTris buffer (pH 7.4) that contains 100 mmKCl at 23 C in a microfluidic chamber. Unfolding events, indicated by a sudden change in contour length (L) during mechanical stretching, were recorded in forceextension (FX) curves (Physique1 B). Using probability density distribution of Land Firocoxib bootstrap statistical analyses (PoDNano,[23] see the Supporting Information), the Lhistograms were deconvoluted into two major populations with Gaussian centers at 9.40.2 and 5.70.3 nm (Figure1 Binset). Consistent with earlier reports,[24] these Lpopulations represent a GQ structure (9.4 nm) and partially folded species (5.7 nm). The latter may assume a G-Triplex[25,26] conformation. Comparable Lpopulations were observed when the TERRA-G4 was incubated with the small-molecule ligand cPDS or the antibody BG4 (Physique2, bottom panels), which implies that these binding.