Sequence and Solvent Effects on Telomeric DNA Bimolecular G‑Quadruplex Folding Kinetics

Telomeric DNA sequences are particularly polymorphic: the adopted structure is exquisitely sensitive to the sequence and to the chemical environment, for example, solvation. Dehydrating conditions are known to stabilize G-quadruplex structures, but information on how solvation influences the individual rates of folding and unfolding of G-quadruplexes remains scarce. Here, we used electrospray mass spectrometry for the first time to monitor bimolecular G-quadruplex formation from 12-mer telomeric strands, in the presence of common organic cosolvents (methanol, ethanol, isopropanol, and acetonitrile). Based on the ammonium ion distribution, the total dimer signal was decomposed into contributions from the parallel and antiparallel structures to obtain individual reaction rates, and the antiparallel G-quadruplex structure was found to form faster than the parallel one. A dimeric reaction intermediate, in rapid equilibrium with the single strands, was also identified. Organic cosolvents increase the stability of the final structures mainly by increasing the <i>folding</i> rates. Our quantitative analysis of reaction rate dependence on cosolvent percentage shows that organic cosolvent molecules can be captured or released upon G-quadruplex formation, highlighting that they are not inert with DNA. In contrast to the folding rates, the G-quadruplex <i>unfolding</i> rates are almost insensitive to solvation effects, but are instead governed by the sequence and by the final structure: parallel dimers dissociate slower than antiparallel dimers only when thymine bases are present at the 5′-end. These results contribute unraveling the folding pathways of telomeric G-quadruplexes. The solvent effects revealed here enlighten that G-quadruplex structure in dehydrated, and molecularly crowded environments are modulated by the nature of cosolvent (e.g., methanol favors antiparallel structures) due to direct interactions, and by the time scale of the reaction, with >200-fold acceleration of bimolecular G-quadruplex formation in the presence of 60% cosolvent

UV Spectroscopy of DNA Duplex and Quadruplex Structures in the Gas Phase

UV absorption spectroscopy is one of the most widely used methods to monitor nucleic acid folding in solution, but the absorption readout is the weighted average contribution of all species present in solution. Mass spectrometry, on the other hand, is able to separate constituents of the solution based on their mass, but methods to probe the structure of each constituent are needed. Here, we explored whether gas-phase UV spectroscopy can give an indication of DNA folding in ions isolated by electrospray mass spectrometry. Model DNA single strands, duplexes, and G-quadruplexes were extracted from solution by electrospray; the anions were stored in a quadrupole ion trap and irradiated by a tunable laser to obtain the UV action spectra of each complex. We found that the duplex and quadruplex spectra are significantly different from the spectra of single strands, thereby suggesting that electronic spectroscopy can be used to probe the DNA gas-phase structure and obtain information about the intrinsic properties of high-order DNA structure

Specific Stabilization of <i>c‑MYC</i> and <i>c‑KIT</i> G-Quadruplex DNA Structures by Indolylmethyleneindanone Scaffolds

Stabilization of G-quadruplex DNA structures by small molecules has emerged as a promising strategy for the development of anticancer drugs. Since G-quadruplex structures can adopt various topologies, attaining specific stabilization of a G-quadruplex topology to halt a particular biological process is daunting. To achieve this, we have designed and synthesized simple structural scaffolds based on an indolylmethyleneindanone pharmacophore, which can specifically stabilize the parallel topology of promoter quadruplex DNAs (<i>c-MYC</i>, <i>c-KIT1</i>, and <i>c-KIT2</i>), when compared to various topologies of telomeric and duplex DNAs. The lead ligands (<b>InEt2</b> and <b>InPr2</b>) are water-soluble and meet a number of desirable criteria for a small molecule drug. Highly specific induction and stabilization of the <i>c-MYC</i> and <i>c-KIT</i> quadruplex DNAs (Δ<i>T</i>_1/2 up to 24 °C) over telomeric and duplex DNAs (Δ<i>T</i>_1/2 ∼ 3.2 °C) by these ligands were further validated by isothermal titration calorimetry and electrospray ionization mass spectrometry experiments (<i>K</i>_a ∼ 10⁵ to 10⁶ M^–1). Low IC₅₀ (∼2 μM) values were emerged for these ligands from a <i>Taq</i> DNA polymerase stop assay with the <i>c-MYC</i> quadruplex forming template, whereas the telomeric DNA template showed IC₅₀ values >120 μM. Molecular modeling and dynamics studies demonstrated the 5′- and 3′-end stacking modes for these ligands. Overall, these results demonstrate that among the >1000 quadruplex stabilizing ligands reported so far, the indolylmethyleneindanone scaffolds stand out in terms of target specificity and structural simplicity and therefore offer a new paradigm in topology specific G-quadruplex targeting for potential therapeutic and diagnostic applications

Structure of Triplex DNA in the Gas Phase

Extensive (more than 90 microseconds) molecular dynamics simulations complemented with ion-mobility mass spectrometry experiments have been used to characterize the conformational ensemble of DNA triplexes in the gas phase. Our results suggest that the ensemble of DNA triplex structures in the gas phase is well-defined over the experimental time scale, with the three strands tightly bound, and for the most abundant charge states it samples conformations only slightly more compact than the solution structure. The degree of structural alteration is however very significant, mimicking that found in duplex and much larger than that suggested for G-quadruplexes. Our data strongly supports that the gas phase triplex maintains an excellent memory of the solution structure, well-preserved helicity, and a significant number of native contacts. Once again, a linear, flexible, and charged polymer as DNA surprises us for its ability to retain three-dimensional structure in the absence of solvent. Results argue against the generally assumed roles of the different physical interactions (solvent screening of phosphate repulsion, hydrophobic effect, and solvation of accessible polar groups) in modulating the stability of DNA structures

Unexpected Position-Dependent Effects of Ribose G‑Quartets in G‑Quadruplexes

To understand the role of ribose G-quartets and how they affect the properties of G-quadruplex structures, we studied three systems in which one, two, three, or four deoxyribose G-quartets were substituted with ribose G-quartets. These systems were a parallel DNA intramolecular G-quadruplex, d­(TTGGG­TGGGT­TGGGT­GGGTT), and two tetramolecular G-quadruplexes, d­(TGGGT) and d­(TGGGGT). Thermal denaturation experiments revealed that ribose G-quartets have position-dependent and cumulative effects on G-quadruplex stability. An unexpected <i>destabilization</i> was observed when rG quartets were presented at the 5′-end of the G stack. This observation challenges the general belief that RNA residues stabilize G-quadruplexes. Furthermore, in contrast to past proposals, hydration is not the main factor determining the stability of our RNA/DNA chimeric G-quadruplexes. Interestingly, the presence of rG residues in a central G-quartet facilitated the formation of additional tetramolecular G-quadruplex topologies showing positive circular dichroism signals at 295 nm. 2D NMR analysis of the tetramolecular TGgGGT (lowercase letter indicates ribose) indicates that Gs in the 5′-most G-quartet adopt the <i>syn</i> conformation. These analyses highlight several new aspects of the role of ribose G-quartets on G-quadruplex structure and stability, and demonstrate that the positions of ribose residues are critical for tuning G-quadruplex properties

Molecular Insights into O‑Linked Sialoglycans Recognition by the Siglec-Like SLBR‑N (SLBR_UB10712) of Streptococcus gordonii

Streptococcus gordonii is a Gram-positive bacterial species that typically colonizes the human oral cavity, but can also cause local or systemic diseases. Serine-rich repeat (SRR) glycoproteins exposed on the S. gordonii bacterial surface bind to sialylated glycans on human salivary, plasma, and platelet glycoproteins, which may contribute to oral colonization as well as endocardial infections. Despite a conserved overall domain organization of SRR adhesins, the Siglec-like binding regions (SLBRs) are highly variable, affecting the recognition of a wide range of sialoglycans. SLBR-N from the SRR glycoprotein of S. gordonii UB10712 possesses the remarkable ability to recognize complex core 2 O-glycans. We here employed a multidisciplinary approach, including flow cytometry, native mass spectrometry, isothermal titration calorimetry, NMR spectroscopy from both protein and ligand perspectives, and computational methods, to investigate the ligand specificity and binding preferences of SLBR-N when interacting with mono- and disialylated core 2 O-glycans. We determined the means by which SLBR-N preferentially binds branched α2,3-disialylated core 2 O-glycans: a selected conformation of the 3′SLn branch is accommodated into the main binding site, driving the sTa branch to further interact with the protein. At the same time, SLBR-N assumes an open conformation of the CD loop of the glycan-binding pocket, allowing one to accommodate the entire complex core 2 O-glycan. These findings establish the basis for the generation of novel tools for the detection of specific complex O-glycan structures and pave the way for the design and development of potential therapeutics against streptococcal infections

Molecular Insights into O‑Linked Sialoglycans Recognition by the Siglec-Like SLBR‑N (SLBR_UB10712) of Streptococcus gordonii

Streptococcus gordonii is a Gram-positive bacterial species that typically colonizes the human oral cavity, but can also cause local or systemic diseases. Serine-rich repeat (SRR) glycoproteins exposed on the S. gordonii bacterial surface bind to sialylated glycans on human salivary, plasma, and platelet glycoproteins, which may contribute to oral colonization as well as endocardial infections. Despite a conserved overall domain organization of SRR adhesins, the Siglec-like binding regions (SLBRs) are highly variable, affecting the recognition of a wide range of sialoglycans. SLBR-N from the SRR glycoprotein of S. gordonii UB10712 possesses the remarkable ability to recognize complex core 2 O-glycans. We here employed a multidisciplinary approach, including flow cytometry, native mass spectrometry, isothermal titration calorimetry, NMR spectroscopy from both protein and ligand perspectives, and computational methods, to investigate the ligand specificity and binding preferences of SLBR-N when interacting with mono- and disialylated core 2 O-glycans. We determined the means by which SLBR-N preferentially binds branched α2,3-disialylated core 2 O-glycans: a selected conformation of the 3′SLn branch is accommodated into the main binding site, driving the sTa branch to further interact with the protein. At the same time, SLBR-N assumes an open conformation of the CD loop of the glycan-binding pocket, allowing one to accommodate the entire complex core 2 O-glycan. These findings establish the basis for the generation of novel tools for the detection of specific complex O-glycan structures and pave the way for the design and development of potential therapeutics against streptococcal infections

Molecular Insights into O‑Linked Sialoglycans Recognition by the Siglec-Like SLBR‑N (SLBR_UB10712) of Streptococcus gordonii

Streptococcus gordonii is a Gram-positive bacterial species that typically colonizes the human oral cavity, but can also cause local or systemic diseases. Serine-rich repeat (SRR) glycoproteins exposed on the S. gordonii bacterial surface bind to sialylated glycans on human salivary, plasma, and platelet glycoproteins, which may contribute to oral colonization as well as endocardial infections. Despite a conserved overall domain organization of SRR adhesins, the Siglec-like binding regions (SLBRs) are highly variable, affecting the recognition of a wide range of sialoglycans. SLBR-N from the SRR glycoprotein of S. gordonii UB10712 possesses the remarkable ability to recognize complex core 2 O-glycans. We here employed a multidisciplinary approach, including flow cytometry, native mass spectrometry, isothermal titration calorimetry, NMR spectroscopy from both protein and ligand perspectives, and computational methods, to investigate the ligand specificity and binding preferences of SLBR-N when interacting with mono- and disialylated core 2 O-glycans. We determined the means by which SLBR-N preferentially binds branched α2,3-disialylated core 2 O-glycans: a selected conformation of the 3′SLn branch is accommodated into the main binding site, driving the sTa branch to further interact with the protein. At the same time, SLBR-N assumes an open conformation of the CD loop of the glycan-binding pocket, allowing one to accommodate the entire complex core 2 O-glycan. These findings establish the basis for the generation of novel tools for the detection of specific complex O-glycan structures and pave the way for the design and development of potential therapeutics against streptococcal infections