Intermediates Stabilized by Tryptophan Pairs Exist in Trpzip Beta-Hairpins

Transitions of protein secondary structures, such as alpha-helices and beta-hairpins, are often too small and too fast to follow by many single-molecular approaches. Here we describe new population deconvolution methods to investigate the mechanical unfolding/refolding events in Trpzip β-hairpins that are tethered between two optically trapped polystyrene particles through click chemistry. The application of force to the Trpzip peptides shifted population distribution, which allowed us to identify intermediates from regular force–extension curves of the peptides after population deconvolution analysis. Comparison of the intermediates between the Trpzip2 and Trpzip4 peptides suggests the intermediates are likely stabilized by the tryptophan pair stacking. We anticipate the method of population deconvolution described here can offer a unique capability to investigate fast transitions in small biological structures

Quantification of Chemical and Mechanical Effects on the Formation of the G‑Quadruplex and i‑Motif in Duplex DNA

The formation of biologically significant tetraplex DNA species, such as G-quadruplexes and i-motifs, is affected by chemical (ions and pH) and mechanical [superhelicity (σ) and molecular crowding] factors. Because of the extremely challenging experimental conditions, the relative importance of these factors on tetraplex folding is unknown. In this work, we quantitatively evaluated the chemical and mechanical effects on the population dynamics of DNA tetraplexes in the insulin-linked polymorphic region using magneto-optical tweezers. By mechanically unfolding individual tetraplexes, we found that ions and pH have the largest effects on the formation of the G-quadruplex and i-motif, respectively. Interestingly, superhelicity has the second largest effect followed by molecular crowding conditions. While chemical effects are specific to tetraplex species, mechanical factors have generic influences. The predominant effect of chemical factors can be attributed to the fact that they directly change the stability of a specific tetraplex, whereas the mechanical factors, superhelicity in particular, reduce the stability of the competing species by changing the kinetics of the melting and annealing of the duplex DNA template in a nonspecific manner. The substantial dependence of tetraplexes on superhelicity provides strong support that DNA tetraplexes can serve as topological sensors to modulate fundamental cellular processes such as transcription

Mechanochemical Sensing of Single and Few Hg(II) Ions Using Polyvalent Principles

Sensitivity of biosensors is set by the dissociation constant (<i>K</i>_d) between analytes and probes. Although potent amplification steps can be accommodated between analyte recognition and signal transduction in a sensor to improve the sensitivity 4–6 orders of magnitude below <i>K</i>_d, they compromise temporal resolution. Here, we demonstrated mechanochemical sensing that broke the <i>K</i>_d limit by 9 orders of magnitude for Hg detection without amplifications. Analogous to trawl fishing, we introduced multiple Hg binding units (thymine (T)–T pairs) in a molecular trawl made of two poly-T strands. Inspired by dipsticks to gauge content levels, mechanical information (force/extension) of a DNA hairpin dipstick was used to measure the single or few Hg²⁺ ions bound to the molecular trawl, which was levitated by two optically trapped particles. The multivalent binding and single-molecule sensitivity allowed us to detect unprecedented 1 fM Hg ions in 20 min in field samples treated by simple filtrations

Quantification of Topological Coupling between DNA Superhelicity and G‑quadruplex Formation

It has been proposed that new transcription modulations can be achieved via topological coupling between duplex DNA and DNA secondary structures, such as G-quadruplexes, in gene promoters through superhelicity effects. Limited by available methodologies, however, such a coupling has not been quantified directly. In this work, using novel magneto-optical tweezers that combine the nanometer resolution of optical tweezers and the easy manipulation of magnetic tweezers, we found that the flexibility of DNA increases with positive superhelicity (σ). More interestingly, we found that the population of G-quadruplex increases linearly from 2.4% at σ = 0.1 to 12% at σ = −0.03. The population then rapidly increases to a plateau of 23% at σ < −0.05. The rapid increase coincides with the melting of double-stranded DNA, suggesting that G-quadruplex formation is correlated with DNA melting. Our results provide evidence for topology-mediated transcription modulation at the molecular level. We anticipate that these high-resolution magneto-optical tweezers will be instrumental in studying the interplay between the topology and activity of biological macromolecules from a mechanochemical perspective

A Mechanosensor Mechanism Controls the G‑Quadruplex/i-Motif Molecular Switch in the <i>MYC</i> Promoter NHE III₁

<i>MYC</i> is overexpressed in many different cancer types and is an intensively studied oncogene because of its contributions to tumorigenesis. The regulation of <i>MYC</i> is complex, and the NHE III₁ and FUSE elements rely upon noncanonical DNA structures and transcriptionally induced negative superhelicity. In the NHE III₁ only the G-quadruplex has been extensively studied, whereas the role of the i-motif, formed on the opposite C-rich strand, is much less understood. We demonstrate here that the i-motif is formed within the 4CT element and is recognized by hnRNP K, which leads to a low level of transcription activation. For maximal hnRNP K transcription activation, two additional cytosine runs, located seven bases downstream of the i-motif-forming region, are also required. To access these additional runs of cytosine, increased negative superhelicity is necessary, which leads to a thermodynamically stable complex between hnRNP K and the unfolded i-motif. We also demonstrate mutual exclusivity between the <i>MYC</i> G-quadruplex and i-motif, providing a rationale for a molecular switch mechanism driven by SP1-induced negative superhelicity, where relative hnRNP K and nucleolin expression shifts the equilibrium to the on or off state

CD experiments of ILPR-I3 at different pH and temperature in a 10 mM sodium phosphate buffer with 100 mM KCl and 5 µM DNA concentration.

<p>(A) CD spectra of the ILPR-I3 in pH 4.5–8.0. (B) Peak wavelength <i>vs</i> pH for the ILPR-I3 (obtained from (A)) and ILPR-I4 (obtained from the CD spectra of the ILPR-I4 at pH 4.5–8.0, data not shown). (C) CD spectra acquired at 23–68°C (pH 5.5). (D) Peak wavelength <i>vs</i> temperature (obtained from (C)). The transition points in B) and D) are determined by sigmoidal fitting (solid curves).</p

Sequences of wild type ILPR-I4 and ILPR-I3, a scrambled sequence, and the mutants used in this study.

<p>Sequences of wild type ILPR-I4 and ILPR-I3, a scrambled sequence, and the mutants used in this study.</p

Mutation analysis in a 10 mM sodium phosphate buffer (pH 5.5) with 100 mM KCl.

<p>(A) 295 nm UV melting curves of the ILPR-I3 (“Wild Type”) and the mutants at 10 µM concentration. (B) Top panel, <i>T</i>_1/2-melt of the mutants and the ILPR-I3. “W” depicts the wild type ILPR-I3. Bottom panel, CD peak shift of the mutants and the scrambled sequence (ILPR-S3) with respect to the 285 nm peak in the ILPR-I3. The horizontal dotted lines (green) represent the average value for each C4 tract. Statistical treatment is represented by the <i>P</i> values in the bottom panel. Please refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039271#pone-0039271-t001" target="_blank">Table 1</a> for DNA sequences.</p

Mutually Exclusive Formation of G‑Quadruplex and i‑Motif Is a General Phenomenon Governed by Steric Hindrance in Duplex DNA

G-Quadruplex and i-motif are tetraplex structures that may form in opposite strands at the same location of a duplex DNA. Recent discoveries have indicated that the two tetraplex structures can have conflicting biological activities, which poses a challenge for cells to coordinate. Here, by performing innovative population analysis on mechanical unfolding profiles of tetraplex structures in double-stranded DNA, we found that formations of G-quadruplex and i-motif in the two complementary strands are mutually exclusive in a variety of DNA templates, which include human telomere and promoter fragments of hINS and hTERT genes. To explain this behavior, we placed G-quadruplex- and i-motif-hosting sequences in an offset fashion in the two complementary telomeric DNA strands. We found simultaneous formation of the G-quadruplex and i-motif in opposite strands, suggesting that mutual exclusivity between the two tetraplexes is controlled by steric hindrance. This conclusion was corroborated in the BCL-2 promoter sequence, in which simultaneous formation of two tetraplexes was observed due to possible offset arrangements between G-quadruplex and i-motif in opposite strands. The mutual exclusivity revealed here sets a molecular basis for cells to efficiently coordinate opposite biological activities of G-quadruplex and i-motif at the same dsDNA location

Formation of an intermolecular i-motif.

<p>(A) Schematic of the formation of an intermolecular i-motif. The proposed structure in the ILPR-I3 is shown on the left. Each C:CH⁺ pair is represented by two opposite rectangles. (B) PAGE gel image of the Br₂ footprinting experiment. Lane 1, the ILPR-I3/ILPR-I1 (I₃+I₁) mixture at pH 7.0. Lane 2, the I₃+I₁ sample at pH 5.5. Lane 3, the ILPR-I3 (I₃) at pH 5.5. Lane 4, the ILPR-I4 (I₄) at pH 5.5. The band intensity for lane 2 is shown to the left of the gel. The fold protection for the I₃+I₁ sample at pH 5.5 is shown to the right. The dotted vertical lines indicate the average fold protection for each C4 tract. The blue arrows indicate the loop cytosines. Error bar represents the standard deviation of three independent experiments. The blue arrows indicate the cytosines in the ACA section of each fragment. Note that the fold protection for adenines at 3'-end (indicated by asterisk *) is not accurate since they are close to the uncut oligo. (C) Normalized rupture force histogram for the I₃+I₁ sample at pH 5.5. The solid black curve represents a two-peak Gaussian function. The dotted curve is the Gaussian fit for the rupture force histogram of the ILPR-I3 at pH 5.5.</p