Understanding the topology adopted by individual G-quadruplex (GQ)-forming sequences in vivo and targeting a specific GQ motif among others in the genome will have a profound impact on GQ-directed therapeutic strategies. However, this remains a major challenge as most of the tools poorly distinguish different GQ conformations and are not suitable for both cell-free and in-cell analysis. Here, we describe an innovative probe design to investigate GQ conformations and recognition in both cell-free and native cellular environments by using a conformation-sensitive dual-app nucleoside analogue probe. The nucleoside probe, derived by conjugating fluorobenzofuran at the 5-position of 2'-deoxyuridine, is composed of a microenvironment-sensitive fluorophore and an in-cell NMR compatible F label. This noninvasive nucleoside, incorporated into the human telomeric DNA oligonucleotide repeat, serves as a common probe to distinguish different GQ topologies and quantify topology-specific binding of ligands by fluorescence and NMR techniques. Importantly, unique signatures displayed by theF-labeled nucleoside for different GQs enabled a systematic study in Xenopus laevis oocytes to provide new structural insights into the GQ topologies adopted by human telomeric overhang in cells, which so far has remained unclear. Studies using synthetic cell models, immunostaining on fixed cells, and crystallization conditions suggest that parallel GQ is the preferred conformation of telomeric DNA repeat. However, our findings using the dual-app probe clearly indicate that multiple structures including hybrid-type parallel-antiparallel and parallel GQs are formed in the cellular environment. Taken together, our findings open new experimental strategies to investigate topology, recognition, and therapeutic potential of individual GQ-forming motifs in a biologically relevant context.
Biophysical and biochemical investigations provide compelling evidence connecting the four-stranded G-quadruplex (GQ) structure with its role in regulating multiple cellular processes. Hence, modulating the function of GQs by using small molecule binders is being actively pursued as a strategy to develop new chemotherapeutic agents. However, sequence diversity and structural polymorphism of GQs have posed immense challenges in terms of understanding what conformation a G-rich sequence adopts inside the cell and how to specifically target a GQ motif amidst several other GQ-forming sequences. In this context, here we review recent developments in the applications of biophysical tools that use fluorescence readout to probe the GQ structure and recognition in cell-free and cellular environments. First, we provide a detailed discussion on the utility of covalently labeled environment-sensitive fluorescent nucleoside analogs in assessing the subtle difference in GQ structures and their ligand binding abilities. Furthermore, a detailed discussion on structure-specific antibodies and small molecule probes used to visualize and confirm the existence of DNA and RNA GQs in cells is provided. We also highlight the open challenges in the study of tetraplexes (GQ and i-motif structures) and how addressing these challenges by developing new tools and techniques will have a profound impact on tetraplex-directed therapeutic strategies.
The development of biophysical systems that enable an understanding of the structure and ligand‐binding properties of G‐quadruplex (GQ)‐forming nucleic acid sequences in cells or models that mimic the cellular environment would be highly beneficial in advancing GQ‐directed therapeutic strategies. Herein, the establishment of a biophysical platform to investigate the structure and recognition properties of human telomeric (H‐Telo) DNA and RNA repeats in a cell‐like confined environment by using conformation‐sensitive fluorescent nucleoside probes and a widely used cellular model, bis(2‐ethylhexyl) sodium sulfosuccinate reverse micelles (RMs), is described. The 2′‐deoxy and ribonucleoside probes, composed of a 5‐benzofuran uracil base analogue, faithfully report the aqueous micellar core through changes in their fluorescence properties. The nucleoside probes incorporated into different loops of H‐Telo DNA and RNA oligonucleotide repeats are minimally perturbing and photophysically signal the formation of respective GQ structures in both aqueous buffer and RMs. Furthermore, these sensors enable a direct comparison of the binding affinity of a ligand to H‐Telo DNA and RNA GQ structures in the bulk and confined environment of RMs. These results demonstrate that this combination of a GQ nucleoside probe and easy‐to‐handle RMs could provide new opportunities to study and devise screening‐compatible assays in a cell‐like environment to discover GQ binders of clinical potential.
Synthesis of a highly responsive fluorescent ribonucleoside analogue based on a 5-methoxybenzofuran uracil core, enzymatic incorporation of its triphosphate substrate into RNA transcripts, and its utility in the specific detection and estimation of Hg2+-ion-mediated metallo-base pair formation in DNA–RNA and RNA–RNA duplexes are described.
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