Probing G-Quadruplexes Conformational Dynamics and Nano-Mechanical Interactions at the Single Molecule Level: Techniques and Perspectives

Abstract

The analysis of nucleic acid structures, topologies, nano-mechanics and interactions with ligands and other biomacromolecules (most notably proteins) at the single molecule level has become a fundamental topic in molecular biophysics over the last two decades. Techniques such as molecular tweezers, single-molecule fluorescence resonance energy transfer, and atomic force microscopy have enabled us to disclose an unprecedented insight into the mechanisms governing gene replication, transcription and regulation. In this minireview, we survey the main working principles and discuss technical caveats of the above techniques, using as a fil-rouge the history of their achievements in dissecting G-quadruplexes. The revised literature offers a clear example of the superior ability of single-molecule techniques with respect to ensemble techniques to unveil the structural and functional diversity of the several polymorphs corresponding to a single G-quadruplex folding sequence, thus shedding new light on the extreme complexity of these fascinating non-Watson–Crick structures.

1. Introduction

G-quadruplexes (G4s) are non-canonical secondary structures of nucleic acids that can occur at sequences containing at least four runs of consecutive guanines. At the genomic level, these motifs are significantly enriched at some functional sites, such as telomeres, promoters, and 5’-UTR domains. In addition, they appear to be significantly conserved throughout evolution. At first, G4 functional roles were addressed at telomeres where long single-stranded G-rich domains are present. Here, their G4 folding was found to impair the telomerase elongation activity. Later on, bioinformatic, in vitro and in-cell studies highlighted their occurrence at multiple genomic sites with an evolutionarily conserved enrichment at the promoter level, thus pointing them as switches for transcription. In particular, at these positions, the G4 formation has been proven to be able to alter the transcription factors recruitment [1]. Interestingly, for several oncogenes (MYC, KIT, BLC2, etc.), such a mechanism was found to cause a reduction in the protein expression level, although alternative patterns are not excluded [2]. These data, along with several in-cell and in vivo studies, extensively confirm their fundamental roles in many cellular regulatory processes [3]. This scenario is fully consistent with their exploitation as targets in oncology, neurological disorders and, more recently, antiviral treatments. The extreme versatility of G4s in acting as signaling elements in metabolic pathways [4,5,6,7] largely depends on their conformational polymorphism. First, it is worth noting that, from a structural point of view, the key architectural element of G4s is the pairing of four guanines through Hoogsteen hydrogen bonds, which drives the formation of a tetra-helical arrangement. Consequently, their features are distinct from the more common double helix. Moreover, in G4s, the four strands can be arranged with different mutual orientations and, in the monomeric structures, they are interconnected by three loops that can vary in terms of length and composition. Finally, different G4s can assume multiple topological arrangements [8,9,10], and even the same sequence can fold into different structures [11,12].

The full landscape of these complex structural equilibria depends on environmental conditions [13,14], exertion of forces and/or torques on DNA by proteins [15,16,17] and interaction with ligands [18,19,20,21,22]. This latter point is of particular interest due to the fact that such ligands may tune the biological functions of the targeted G4, and thus are promising to act as novel therapeutics for several diseases [1,23,24,25,26,27,28].

From this picture, it is easy to derive potential intriguing correlations between the molecular features of G4s and a wealth of fundamental aspects of cell life, such as metabolism, self-regulation, senescence, survival, and pathological pathways. For these reasons, it is of pivotal importance to set up biophysical tools capable of yielding detailed information about the conformational dynamics of G4-folding in DNA and RNA sequences, as well as probing the effects of nanomechanical forces and torques that mimic those solicitations which are physiologically exerted, e.g., by proteins. Due to the extreme conformational diversity of G4s, samples are expected to present dramatic heterogeneity. As a result, probing the above-mentioned features at the single-molecule level is mandatory to faithfully reconstruct the statistical distributions of either the structures assumed or the nanomechanical interactions acting on a G4-folding sequence. Here, we report on advanced techniques developed for this purpose, review the fundamental literature on the topic, and critically assess their strengths and weaknesses.

2. Conformational Studies — Single-Molecule Fluorescence Resonance Energy Transfer

Historically, biomolecular conformations have been extensively and successfully investigated by exploiting the fluorescence resonance energy transfer (FRET) phenomenon. Theorized in 1949 by T. Förster [29], FRET is a quantum dipole–dipole resonance interaction occurring between two chromophores, in this context called donor (D) and acceptor (A). The fluorescence spectrum of D is partially overlapped with the absorption spectrum of A. Upon illumination in the absorption band of D, the excitation energy can be exchanged between the two dyes, resulting in a reduced fluorescence emission of D and a concomitant promotion of A to an electronically excited state. If A is also a fluorophore, its emission can be observed (see Figure 1b). As the efficiency of the energy transfer drops with the sixth power of the D-to-A distance, FRET can be exploited as a precise “spectroscopic ruler” to measure intramolecular distances in suitably labelled biomolecules [30].

The amount of energy transferred from the D to the A is usually evaluated in terms of the FRET efficiency, which is the ratio between the intensity of the fluorescence emitted by the A to the total fluorescence emitted by A and D (measured in number of photons per unit time):

$$E={\displaystyle \frac{I_{\mathrm{A}}}{I_{\mathrm{A}}+\gamma I_{\mathrm{D}}}}$$

The correction factor $\gamma$ takes into account the different fluorescence quantum yields of the D and the A, as well as different detection efficiencies in the two channels, and depends on the setup and fluorophores used.

At the ensemble level, the FRET technique has been operatively implemented on systems of biological relevance since the early 1970s (for a review of early experiments, see [32,33]). However, the ensemble approach can be tricky in heterogeneous samples [34], where, as a result of conformational polymorphism and dynamics of the labelled biomolecule, the distance between the sites to which the D and the A are attached varies in time as well as from a molecule to another. It is worth noting that in this instance, the limitation of the technique reseeds in the unfeasibility of extrapolating FRET efficiency distributions from the data, which inherently provide only a mean FRET efficiency value consisting in the average over long times (compared with those proper of molecular dynamics) and high numbers of molecules of the actual ones. Implementation of FRET at the single-molecule level dates back to the 1990s [35,36,37,38]. It allows for overcoming the above limitation and provides an invaluable tool to probe the structural heterogeneity of a biomolecular specimen. Indeed, single-molecule FRET (SM-FRET) is nowadays a preferred technique to reveal the number of different structures assumed by a peptide [39,40,41,42,43] or nucleic acid sequence [44], as well as to unravel complex formation with defined partners. Accordingly, it can be optimally exploited to investigate the conformational diversity, dynamics, and transitions of G4s under strictly controlled experimental conditions. It should be noted that, by themselves, SM-FRET data can only yield indirect information on the conformational dynamics of a biomolecule. Indeed, the observable which is directly measured in an SM-FRET experiment is the D-to-A distance, which cannot always be straightforwardly connected with conformational features of the spacer (i.e., the labelled biomolecule of interest). However, with the advent of computer sciences, SM-FRET data can be interpreted in the light of dedicated molecular dynamics simulations, which offer a boosted comprehension by providing the needed insight to bridge the gap between the D-to-A distance datum and the corresponding structural conformation. Exemplary studies have been performed on the topic in the last few years [45,46,47,48]. Pioneering attempts to address the G4 topologies of the folded human telomeric sequence (TTAGGG)4 were made as early as 2003 [49]. Since then, the SM-FRET technique has been systematically applied to probe telomeric G4s under various experimental conditions [14,50,51,52,53,54,55,56,57,58], and it has been extended to additional G4-forming sequences, such as those occurring at gene promoters [59,60,61,62,63,64], in both their wild-type and mutated forms, and in the presence or absence of partner proteins [65].

Currently, two different implementations of SM-FRET exist: total internal reflection microscopy-based (TIRF-FRET) and confocal microscopy-based (CONF-FRET). These methods have been extensively described in excellent papers by Ha [36,66,67,68,69] and applied to characterize G4s. Each method has its advantages and drawbacks. TIRF-FRET allows for real-time monitoring of the conformational dynamics of single DNA molecules tethered to a surface for several seconds, which is relatively long compared to the timescales of conformational transitions [61,62]. In contrast, CONF-FRET, especially in its standard implementation, captures snapshots of molecules freely diffusing in and out of the confocal observation volume [70], thus producing fluorescence bursts (see Figure 1). The conformational distribution of species in the specimen is determined by sampling several of such independent fluorescence burst events. Notably, the time for an oligonucleotide [71,72], oligopeptide [43,73,74], or even a high molecular weight protein [63,75] to cross the observation volume is typically on the order of hundreds of microseconds. Given that the fastest folding–unfolding dynamics and interconversion kinetics between differently folded conformations occur on timescales of hundreds of milliseconds [41,61,62,76], negligible conformational changes are typically assumed during diffusion time.

Furthermore, CONF-FRET is minimally invasive and can monitor system evolution over hours [77], whereas the observation time in TIRF-FRET experiments is inherently limited by dye photobleaching, which typically occurs within tens of seconds [66,78,79,80]. Moreover, the environment experienced by freely diffusing biomolecules in CONF-FRET closely resembles physiological conditions compared to TIRF-FRET experiments, where biomolecules can interact with the surface to which they are tethered. This is particularly critical for oligonucleotide probes, which behave as polyelectrolytes and require surface passivation [66,81]. Additionally, the linking procedure requires careful design of the oligonucleotide construct [67], and the surface must undergo thorough cleaning procedures to minimize background interference [81,82,83]. The photophysics of donor and acceptor chromophores can also be perturbed unpredictably by proximity to a solid surface [84,85].

As to a direct comparison between results yielded by the two SM-FRET variants, to the best of our knowledge, there exists only one instance in which the same G4-forming sequence has been probed by means of both methods. Namely, in 2007, Balasubramanian and collaborators [59] performed a characterization of oligonucleotides mimicking the sequences of the G4s c-kit1 and c-kit2 embedded within the c-KIT oncogene promoter, exploiting both TIRF- and CONF-FRET. Although their findings appear self-consistent from a qualitative standpoint, the FRET efficiency distributions obtained on homologous samples with the two setups are remarkably different. However, it must be noted that the same authors published a similar study on the same G4-folding sequences in 2010 [60], using the CONF-FRET setup only, and they obtained different results with respect to the homologous measurements they had previously published. This fact indicates that many environmental aspects concur in determining the actual folding of a G4. Thus, the difference recorded in the 2007 experiments on the two setups might be reconducted for reasons other than the SM-FRET option chosen (e.g., slight variations in the buffer or temperature).

TIRF-FRET has been more extensively applied than CONF-FRET to probe G4s so far. Besides investigating the conformations and folding/unfolding kinetics of telomeric G4s under various environmental conditions, specifically different concentrations of cations [14,49,50,52,55,57,58], and the role of small ligands and proteins in tuning such equilibria [54,56,62,86,87,88,89,90,91], analyses have been extended to G4s formed within the promoters of several genes [91], including the oncogenes c-KIT [88,90], c-MYC [61,62,82,87,88,92], and PAX9 [88].

However, in recent years, significant attention has been devoted to designing experimental models that more closely resemble the physiological genomic context, where the G4-forming domain is embedded within a more complex elongated framework. It has been found that the presence of either single- or double-stranded DNA tracts located at the ends of the G4-folding sequence significantly affects the conformational equilibrium of the tetrahelical domain [77,93]. This paves the way for SM-FRET studies aimed at dissecting the structural properties of G4s encompassed within oligonucleotide constructs that better mimic the features of the native DNA environment. For this purpose, CONF-FRET setups offer superior flexibility, thus this approach is expected to obtain more attention in the near future.

3. Probing the G4 Mechanical Stability and Mimicking Traction and Torsion Stresses Exerted on G4 by Proteins in Physiological Environment

As discussed above, in addition to the genomic context, the forces exerted by proteins play pivotal roles in inducing the conversion of double-stranded DNA into a G4. Therefore, valuable information about G4 folding can be extracted from their nanomechanical manipulation using single-molecule force spectroscopy techniques [94,95].

3.1. Optical Tweezers

The unfolding free energy landscapes of biomolecular structures can be determined by inducing controlled traction forces using optical tweezers (OT). In an OT setup, radiation pressure allows a microscopic bead to be held in a focused laser beam (Figure 2). In this trapping mode, it is possible to generate spring-like forces ranging from a few piconewtons to nanonewtons on biomolecules properly functionalized with the bead(s) [96,97]. Since the pioneering works of Ashkin [98,99] in the late 1980s, this technique has found wide applications in biophysics [100,101]. In its simpler implementation for DNA experiments, OT involves tethering the free end of the DNA molecule under investigation to a surface and attaching a dielectric bead [102,103,104]. Alternatively, more than one bead can be attached to the opposite ends of a freely diffusing DNA molecule, creating multiple traps and offering more experimental flexibility [105,106,107,108] (see Figure 2). Overall, these beads serve as probes to measure the forces acting on the DNA molecule (e.g., due to interactions with proteins, environmental or structural changes) or to study its mechanical response using the laser beam as a force actuator. In the field of G4 research, optical tweezers (OT) were originally applied to probe the nanomechanical stability of single G4s by determining their folding free energy. Specifically, Mao’s group studied a G-rich sequence present in the insulin-linked polymorphic region (ILPR), a DNA domain of the insulin gene promoter. The G4-folding sequence was inserted between two double-stranded DNA tracts, either as a single-stranded linker [109] or counterfaced by its complementary cytosine-rich sequence [110]. Under acidic conditions, the complementary strand of a G4-forming site can fold into a different non-canonical secondary structure called an I-motif (iM), a tetrahelix supported by intercalated C − C + base pairs. The authors demonstrated that the G4 was more resistant to mechanical denaturation than the iM. However, they also showed that under conditions compatible with the folding of both structures, only one could be formed at a time due to steric hindrance. These results were confirmed by independent investigations by de Messieres [111].

Expanding on the OT technique, Mao’s group applied it to probe the interaction between neighboring G4s within the ILPR [112], pairs of telomeric G4s [113], and later several repeats [114]. Additionally, they elucidated the stabilizing forces of telomeric G4s in both DNA [115,116,117] and RNA [118], interactions with small ligands [113,114,119,120,121,122,123], folding–unfolding equilibria of G4s in the hTERT promoter [124,125], and the effects of crowding agents on G4 stability [126].

Other groups have focused their efforts on analyzing the effects of condensation on the stability of telomeric RNA G4s [127] and assessing G4-protein interactions [128].

Figure 2. Scheme and principle of operation of an optical tweezer. (a) A trapping laser beam is expanded to fill the back aperture of a high-NA microscope objective and focused into the sample, creating a trapping optical field. White light is transmitted through the sample, collected from the objective and sent to a CCD or CMOS camera through a lens to capture images of the focal plane of the objective in the sample. (b) Detail of the geometry of the trapping beam: inside the sample, the focused laser beam produces a force gradient attracting particles with a refractive index larger than that of the medium in the position of the focus. (c) Example of a force spectroscopy measurement employing two optical tweezers to trap and pull two beads connected to a G4 through a pair of dsDNA handles. The handles are connected to the beads through biotin/streptavidin and digoxigenin/anti-digoxigenin links. Note that this kind of experiment can be performed with a single optical tweezer, by fixing one end of the construct under study to the coverslip or to a micropipette. (d) Example of a force-extension measurement performed with the setup depicted in (c): the distance between the ends of the construct under study is changed over time, and the force (a,b) from [129], used under license; (c,d) from [114], © 2024 Elsevier B.V., its licensors, and contributors, used with permission.

Figure 2. Scheme and principle of operation of an optical tweezer. (a) A trapping laser beam is expanded to fill the back aperture of a high-NA microscope objective and focused into the sample, creating a trapping optical field. White light is transmitted through the sample, collected from the objective and sent to a CCD or CMOS camera through a lens to capture images of the focal plane of the objective in the sample. (b) Detail of the geometry of the trapping beam: inside the sample, the focused laser beam produces a force gradient attracting particles with a refractive index larger than that of the medium in the position of the focus. (c) Example of a force spectroscopy measurement employing two optical tweezers to trap and pull two beads connected to a G4 through a pair of dsDNA handles. The handles are connected to the beads through biotin/streptavidin and digoxigenin/anti-digoxigenin links. Note that this kind of experiment can be performed with a single optical tweezer, by fixing one end of the construct under study to the coverslip or to a micropipette. (d) Example of a force-extension measurement performed with the setup depicted in (c): the distance between the ends of the construct under study is changed over time, and the force (a,b) from [129], used under license; (c,d) from [114], © 2024 Elsevier B.V., its licensors, and contributors, used with permission.

3.2. Magnetic Tweezers

A valuable alternative to OT for probing DNA nanomechanics is represented by magnetic tweezers (MT), which are particularly suitable for assessing the role of mechanical stress in the folding/unfolding of G4s. In the case of MT, magnetic fields generated by either permanent magnets or electromagnets are used to induce forces on biomolecules tethered to magnetic particles [130]. Specifically, an MT apparatus appears as an evolution of a traditional microscope (Figure 3). It is basically composed of an imaging system, an image recording system (usually a CCD camera) that tracks the confined Brownian motion of the magnetic beads, and a magnet mounted on a piezoelectric actuator providing translational and rotational degrees of freedom. The movement of the magnet alters the magnetic field in which the beads are immersed, thereby setting them into motion. These forces are transmitted to the biomolecules to which they are tethered. MT lacks the extreme spatial resolution and flexibility of OT, and applying forces exceeding tens of piconewtons is challenging. However, the ability to easily rotate the magnets constitutes a notable advantage of MT over OT, particularly useful in G4 studies. Decoupling the rotation and translation of the magnets allows for simultaneous, independent, and strictly controllable torsional and tensile stress on DNA single molecules [131]. This capability is particularly relevant because negative supercoiling has been widely reported to enhance the transition of double-stranded DNA to G4 structures [132,133,134].

In order to enable the inspection of G4s through MT experiments, a panel of non-trivial biotechnological solutions must be devised. In a typical MT experiment on DNA, the sample is contained in a flow chamber allowing to vary the DNA substrate features in a controlled way. For instance, the pH and ionic strength of the buffer solution can be tuned while measuring the force-extension curve. Additionally, specific ions (most importantly K⁺ in the case of G4s analyses), ligands or proteins can be injected. The flow chamber may consist either of a capillary tube or in the interstitial space between two coverslips sealed with, e.g., parafilm. The sample must be tethered to the chamber’s internal surface. To this end, both chemical (e.g., thiol/silane) and biochemical (mainly digoxygenin/antidigoxygenin) molecular recognition systems have been exploited. The functionalization of the opposite end of the DNA molecules with micrometric-size superparamagnetic beads typically relies on the binding of biotinylated bases with streptavidin, coating the bead. In order to avoid streptavidin adherence to the glass surface of the chamber, after the adhesion of the DNA sample, but prior to their labeling with the beads, the chamber walls must be passivated. One standard solution is incubation with bovine serum albumin. In experiments in which the G4s are submitted to traction forces, the G4-folding tract is encompassed within two several kbase long double-stranded flanking ends, connecting it to the chamber surface and to the magnetic bead, respectively. Since such specimen is not torsionally constrained, to inspect the double-strand to G4 transition and equilibrium under exertion of external torques double-stranded samples encoding a central tract replicating one or more adjacent sequences capable of folding into G4s have been devised.

MT has been extensively exploited in the last decade to probe the conformational stability of telomeric [136,137] and promoter [137,138,139,140] G4s. Interestingly, MT has proven particularly effective in studies focused on G4s embedded in gene promoters. In vivo, these G4s are counteracted by a complementary strand and exist in equilibrium with double-stranded structures. Besides specific ion abundance, the G4/double-strand equilibrium is primarily dictated by superhelicity, which is locally regulated in vivo by proteins such as helicases or gyrases. MT allow mimicking the effects of such proteins and studying the double-helix to G4 transition under different superhelicity conditions induced by controlled torques applied through magnets onto magnetic beads [140].

Several studies aimed to rationalize or establish benchmarks in the folding pathways of G4s. Specifically, the precise measurement of the work exerted by the magnetic field on the bead to unfold a G4 enabled the reconstruction of unfolding energy landscapes and folding pathways of telomeric G4s [141], as well as the measurement of unfolding energies of telomeric G4s [142] and G4s embedded in the KIT oncogene promoter [140]. The folding kinetics and dynamics of various G4 structures [136,137,138,139] were monitored. Moreover, Cheng and colleagues systematically studied superior mechanical stability and slower unfolding rates of parallel versus antiparallel and hybrid G4s [137,143]. The same group also investigated the role of bulges in tuning folding pathways of typical parallel G4s through the formation of folding intermediates upon the gradual release of tension forces [143]. MT has shown particular versatility also in probing the effects of helicases on the stability of G4s [144], as well as the torques exerted by these proteins on DNA upon binding to G4 structures [145]. The effects of the binding of cisplatin on the folding kinetics of telomeric G4s have also been evaluated with MT [146].

Finally, the role of G4–G4 interactions in tuning the folding equilibrium of both telomeric [147] and promoter [140] G4s has also been recently addressed.

In several instances, MT and OT have been implemented together to combine spatial resolution with the ability to exert torques [133,134,148]. Moreover, to enhance the sensitivity of force-spectroscopy assays regarding different conformational arrangements of the investigated G4s, both MT [149] and OT [150,151] have been performed concurrently with SM-FRET.

Additionally, the MT technique has been used in combination with electron microscopy to demonstrate the compaction of single-stranded telomeric repeats into a bead-like structure upon G4 formation [152].

3.3. Atomic Force Microscopy

Atomic force microscopy (AFM) is a third technique that allows the exertion of pico- to nanonewton forces on single biomolecules, including DNA. The working principle of the atomic force microscope involves monitoring the interaction of a cantilever with the sample surface. As illustrated in Figure 4, this interaction causes tiny flexions of the cantilever tip, which can be detected by measuring the deflections of a laser beam focused on the cantilever. The cantilever can be moved across a sample deposited on a smooth surface (usually a mica layer) in a horizontal plane, using the raster-scanning mode. This mode provides a sub-nanometer resolution map of the surface roughness, giving topological information about the sample [153].

Alternatively, AFM allows the exertion of traction forces up to nanonewtons on objects deposited onto a smooth surface when the cantilever is operated in tapping mode. In tapping mode, scanning a sample surface entails lowering the tip onto the surface sequentially to apply controlled pressure, followed by lifting the cantilever either at a constant velocity or by applying a constant pulling force. The surface is scanned (fishing mode), searching for deposited objects. When the cantilever tip encounters an object, the contact is detected as a change in pressure. Moreover, the stiffness of the obstacle can be measured based on the tip bending at different applied forces.

For the purpose of this review, it is worth noting that even single nucleic acid or peptide molecules fixed onto a mica surface can be “fished” by the cantilever. In this case, adhesion forces cause one end of the biopolymer to bind to the cantilever tip. Consequently, controlled work can be exerted on single molecules by lifting the cantilever at a constant force. Such work can disrupt intramolecular interactions responsible for folding, thereby precisely determining the unfolding energetics [153,157].

In such force-spectroscopy modality, AFM has been applied to probe the stability of either telomeric [158,159] or aptameric [160] G4s, and their binding force to proteins [160]. The technique also allowed the authors to quantify the G4-stabilizing effect of a drug, namely a telomerase inhibitor [156].

On the other hand, AFM has been used also in the imaging mode to certify the formation of G4s in different DNA constructs at varying environmental properties. To the best of our knowledge, the first attempt in this sense was made by Thalhammer and coworkers as early as 2004 [161]. The authors unraveled the interaction of model palindromic G-rich oligonucleotides leading to disruption of hairpins and the formation of a tetrameric G4. Later, similar experiments were conducted by Oliveira-Brett et al. on different sequences at varying cation concentrations in the reaction buffer [162]. In 2009, Edwardson et al. managed to visualize by AFM imaging the formation of a promotorial G4 within a fragment of murine Sg3 switch region amplified from a plasmid [163]. Moreover, the authors thoroughly characterized the morphology of the counterfacing DNA trait, unraveling four different topological arrangements. In a subsequent study [164], some of them also probed the interactions of the same DNA specimen with the nuclear protein PARP-1 and the antibody HF1. Furthermore, they have shown that small ligands can tune in either sense of these interactions. Another study by Wang et al. [165] probed the distribution of folded G4s in a realistic mimic of the telomeric single-strand tail, namely an oligonucleotide sequence consisting of sixteen repeats of the GGGTTA motif. The authors discovered that, on average, only two out of the four possible G4 structures that could theoretically form coexist. Additionally, POT1 was shown to disrupt pre-folded G4s more efficiently than antisense oligonucleotides.

Finally, in a series of studies, Endo and collaborators exploited the DNA-origami technique combined with fast-scanning AFM imaging to monitor in real time the formation of an interstrand G4 with the telomeric repeat [166,167] and the interconversion kinetics between the double-stranded structure and a bubble stabilized by concomitant formation of a G4 counterfaced by an I-motif [168].

4. Conclusions

In this mini-review, we described the main single-molecule biophysical techniques that were exploited to probe the conformational dynamics, folding energetics, binding interactions, and reactivity to nanomechanical stresses of G4-forming sequences.

We revised the main literature on the topic. The milestones of this literature are summarized in Figure 5. Based on this excursus, we critically examined the strengths and pitfalls of each technique. The resulting pattern suggests that a single-molecule approach for the study of G4 folding equilibria, dynamics, and energetics is indispensable to grasp the essence of related biology and offers valuable information for new-conception pharmacological strategies based on the selective targeting of these fascinating secondary structures. At the same time, the combination of several single-molecule methods with more consolidated and standardized biochemical assays as well as state-of-art whole genome and whole-organism investigation protocols is mandatory to achieve a full comprehension of physiological and pathological structure–function relationships and in-cell interactions of G4.

In conclusion, although the single-molecule approach has been extensively and successfully applied to the elucidation of the folding features of selected guanine-rich sequences, mainly the telomeric one and those corresponding to a panel of G4s forming within the promotors of a few oncogenes, wide avenues remain to be explored in this field of investigation, including extending the analysis to a multitude of other sequences, each of those behaving uniquely and being a fundamental piece of the metabolic puzzle. The main frontier remaining, however, is the design of specimens which, whilst lending themselves to single-molecule analysis, mimic the physiological milieu of the G4-forming sequences under quest with increasing reliability and detail.