18 research outputs found
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 ÎČ-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 forceâextension
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 GâQuadruplex and iâMotif 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 (Ï) 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><sub>d</sub>) between analytes and probes. Although potent
amplification steps can be accommodated between analyte recognition
and signal transduction in a sensor to improve the sensitivity 4â6
orders of magnitude below <i>K</i><sub>d</sub>, they compromise
temporal resolution. Here, we demonstrated mechanochemical sensing
that broke the <i>K</i><sub>d</sub> limit by 9 orders of
magnitude for Hg detection without amplifications. Analogous to trawl
fishing, we introduced multiple Hg binding units (thymine (T)âT
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 Hg<sup>2+</sup> 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 Gâquadruplex 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 (Ï).
More interestingly, we found that the population of G-quadruplex increases
linearly from 2.4% at Ï = 0.1 to 12% at Ï = â0.03.
The population then rapidly increases to a plateau of 23% at Ï
< â0.05. 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 GâQuadruplex/i-Motif Molecular Switch in the <i>MYC</i> Promoter NHE III<sub>1</sub>
<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 III<sub>1</sub> and FUSE elements rely upon noncanonical
DNA structures and transcriptionally induced negative superhelicity.
In the NHE III<sub>1</sub> 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 ”M DNA concentration.
<p>(A) CD spectra of the ILPR-I3 in pH 4.5â8.0. (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 4.5â8.0, data not shown). (C) CD spectra acquired at 23â68°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 (âWild Typeâ) and the mutants at 10 ”M concentration. (B) Top panel, <i>T</i><sub>1/2-melt</sub> of the mutants and the ILPR-I3. âWâ 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 GâQuadruplex and iâMotif 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<sup>+</sup> pair is represented by two opposite rectangles. (B) PAGE gel image of the Br<sub>2</sub> footprinting experiment. Lane 1, the ILPR-I3/ILPR-I1 (I<sub>3</sub>+I<sub>1</sub>) mixture at pH 7.0. Lane 2, the I<sub>3</sub>+I<sub>1</sub> sample at pH 5.5. Lane 3, the ILPR-I3 (I<sub>3</sub>) at pH 5.5. Lane 4, the ILPR-I4 (I<sub>4</sub>) at pH 5.5. The band intensity for lane 2 is shown to the left of the gel. The fold protection for the I<sub>3</sub>+I<sub>1</sub> 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 I<sub>3</sub>+I<sub>1</sub> 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