9 research outputs found
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
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><sub>1/2-melt</sub> 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
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
Controlled Particle Collision Leads to Direct Observation of Docking and Fusion of Lipid Droplets in an Optical Trap
As
an intracellular organelle, phospholipid-coated lipid droplets
have shown increasing importance due to their expanding biological
functions other than the lipid storage. The growing biological significance
necessitates a close scrutiny on lipid droplets, which have been proposed
to mature in a cell through processes such as fusion. Unlike phospholipid
vesicles that are well-known to fuse through docking and hemifusion
steps, little is known on the fusion of lipid droplets. Herein, we
used laser tweezers to capture two micrometer-sized 1,2,3-trioleoylglycerol
(triolein) droplets coated with 1-palmitoyl-2-oleoyl-<i>sn</i>-glycero-3-phosphocholine (POPC) that closely resemble intracellular
lipid droplets. We started the fusion processes by a well-controlled
collision between the two lipid droplets in phosphate buffer at pH
7.4. By monitoring the change in the pathway of a trapping laser that
captures the collided lipid droplets, docking and physical fusion
events were clearly distinguished for the first time and their lifetimes
were determined with a resolution of 10 μs after postsynchronization
analysis. Our method revealed that the rate-limiting docking process
is affected by anions according to a Hofmeister series, which sheds
light on the important role of interfacial water shedding during the
process. During the physical fusion, the kinetics between bare triolein
droplets is faster than lipid droplets, suggesting that breaking of
phospholipid coating is involved in the process. This scenario was
further supported by direct observation of a short-lived hemifusion
state with ∼46 ms lifetime in POPC-coated lipid droplets, but
not in bare triolein droplets
Long-Loop G‑Quadruplexes Are Misfolded Population Minorities with Fast Transition Kinetics in Human Telomeric Sequences
Single-stranded guanine (G)-rich sequences at the 3′
end
of human telomeres provide ample opportunities for physiologically
relevant structures, such as G-quadruplexes, to form and interconvert.
Population equilibrium in this long sequence is expected to be intricate
and beyond the resolution of ensemble-average techniques, such as
circular dichroism, NMR, or X-ray crystallography. By combining a
force-jump method at the single-molecular level and a statistical
population deconvolution at the sub-nanometer resolution, we reveal
a complex population network with unprecedented transition dynamics
in human telomeric sequences that contain four to eight TTAGGG repeats.
Our kinetic data firmly establish that G-triplexes are intermediates
to G-quadruplexes while long-loop G-quadruplexes are misfolded population
minorities whose formation and disassembly are faster than G-triplexes
or regular G-quadruplexes. The existence of misfolded DNA supports
the emerging view that structural and kinetic complexities of DNA
can rival those of RNA or proteins. While G-quadruplexes are the most
prevalent species in all the sequences studied, the abundance of a
misfolded G-quadruplex in a particular telomeric sequence decreases
with an increase in the loop length or the number of long-loops in
the structure. These population patterns support the prediction that
in the full-length 3′ overhang of human telomeres, G-quadruplexes
with shortest TTA loops would be the most dominant species, which
justifies the modeling role of regular G-quadruplexes in the investigation
of telomeric structures
Click Chemistry Assisted Single-Molecule Fingerprinting Reveals a 3D Biomolecular Folding Funnel
A 3D folding funnel was proposed in the 1990s to explain
the fast
kinetics exhibited by a biomacromolecule in presence of seemingly
unlimited folding pathways. Over the years, numerous simulations have
been performed with this concept; however, experimental verification
is yet to be attained even for the simplest proteins. Here, we have
used a click chemistry based strategy to introduce six pairs of handles
in a human telomeric DNA sequence. A laser-tweezers-based, single-molecule
structural fingerprinting on the six inter-handle distances reveals
the formation of a hybrid-1 G-quadruplex in the sequence. Kinetic
and thermodynamic fingerprinting on the six trajectories defined by
each handle-pair depict a 3D folding funnel and a kinetic topology
in which the kinetics pertaining to each handle residue is annotated
for this G-quadruplex. We anticipate the methods and the concepts
developed here are well applicable to other biomacromolecules, including
RNA and proteins
Single-Molecule Measurements of the Binding between Small Molecules and DNA Aptamers
Aptamers that bind small molecules can serve as basic
biosensing platforms. Evaluation of the binding constant between an
aptamer and a small molecule helps to determine the effectiveness
of the aptamer-based sensors. Binding constants are often measured
by a series of experiments with varying ligand or aptamer concentrations.
Such experiments are time-consuming, material nonprudent, and prone
to low reproducibility. Here, we use laser tweezers to determine the
dissociation constant for aptamer–ligand interactions at the
single-molecule level from only one ligand concentration. Using an
adenosine 5′-triphosphate disodium salt (ATP) binding aptamer
as an example, we have observed that the mechanical stabilities of
aptamers bound with ATP are higher than those without a ligand. Comparison
of the change in free energy of unfolding (Δ<i><i>G</i></i><sub>unfold</sub>) between these two aptamers
yields a Δ<i><i>G</i></i> of 33 ± 4
kJ/mol for the binding. By applying a Hess-like cycle at room temperature,
we obtained a dissociation constant (<i>K</i><sub>d</sub>) of 2.0 ± 0.2 μM, a value consistent with the <i>K</i><sub>d</sub> obtained from our equilibrated capillary electrophoresis
(CE) (2.4 ± 0.4 μM) and close to that determined by affinity
chromatography in the literature (6 ± 3 μM). We anticipate
that our laser tweezers and CE methodologies may be used to more conveniently
evaluate the binding between receptors and ligands and also serve
as analytical tools for force-based biosensing