Mechanical diversity and folding intermediates of parallel-stranded G-quadruplexes with a bulge

Abstract

A significant number of sequences in the human genome form noncanonical G-quadruplexes (G4s) with bulges or a guanine vacancy. Here, we systematically characterized the mechanical stability of parallel-stranded G4s with a one to seven nucleotides bulge at various positions. Our results show that G4-forming sequences with a bulge form multiple conformations, including fully-folded G4 with high mechanical stability (unfolding forces > 40 pN), partially-folded intermediates (unfolding forces < 40 pN). The folding probability and folded populations strongly depend on the positions and lengths of the bulge. By combining a single-molecule unfolding assay, dimethyl sulfate (DMS) footprinting, and a guanine-peptide conjugate that selectively stabilizes guanine-vacancy-bearing G-quadruplexes (GVBQs), we identified that GVBQs are the major intermediates of G4s with a bulge near the 5′ or 3′ ends. The existence of multiple structures may induce different regulatory functions in many biological processes. This study also demonstrates a new strategy for selectively stabilizing the intermediates of bulged G4s to modulate their functions.

The Cellular Functions and Molecular Mechanisms of G-Quadruplex Unwinding Helicases in Humans

“…In terms of G4s in the human genome, most G4s are noncanonical structures ( Chambers et al, 2015 ), such as bulged G4s ( Zhang Y. et al, 2021 ), long looped G4s ( Agrawal et al, 2014 ), and vacancy G4s ( Li et al, 2015 ; Wang et al, 2020 ). However, there is little research on the interaction of G4 helicases with these structures.…”

Characterization of G-Quadruplexes Folding/Unfolding Dynamics and Interactions with Proteins from Single-Molecule Force Spectroscopy

“…Compared to force-clamp experiments, the force-ramp experiments measure the unfolding force distributions in repeating force-ramp cycles with linearly increased forces (constant loading rate), thus allowing measurement of the unfolding forces of stable G4s ( k u ~10 −6 s −1 ). Another advantage in force-ramp experiments is that for sequences that form multiple structures, the distribution of the unfolding forces and unfolding step sizes can provide information of polymorphism of G4s [ 40 , 62 , 79 ]. Figure 3 B shows an example of force-ramp experiments using MT by changing the magnetic field with time.…”

“…In 2021, Zhang et al further characterized the mechanical stability of noncanonical parallel-stranded bulged G4s. The results showed that bulged G4s form multiple conformations, including fully folded G4s with an unfolding force peak at >40 pN and partially folded intermediates (guanine-vacancy bearing G4s) with an unfolding force peak at <40 pN [ 62 ]. The effects of bulge lengths, bulge positions, and G-vacancies on the folding rates and folding probability have also been systematically analyzed [ 62 ].…”

“…These single-molecule force–spectroscopy techniques have deepened our understanding of the mechanical properties of biomolecules [ 46 ], folding/unfolding dynamics [ 47 , 48 ], and the interactions between biological macromolecules [ 49 ]. In the past two decades, the folding/unfolding dynamics of G4s have been studied by OT [ 50 , 51 , 52 , 53 , 54 ], AFM [ 55 , 56 , 57 , 58 ], and MT [ 59 , 60 , 61 , 62 ]. In vivo, G4s are subjected to various mechanical modifications during transcription and replication, and motor proteins such as polymerases or helicases may exert forces on G4s.…”

Complexity of Guanine Quadruplex Unfolding Pathways Revealed by Atomistic Pulling Simulations

“…Non-telomeric GQs have been investigated, too. It has been shown that GQs with bulges and decreased number of quartets are less stable than complete GQs (72). Conversely, increasing K + concentration has been shown to increase the stability of a three- and four-quartet antiparallel bimolecular GQ (73).…”