4 research outputs found
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
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
Direct Quantification of Loop Interaction and π–π Stacking for G‑Quadruplex Stability at the Submolecular Level
The
well-demonstrated biological functions of DNA G-quadruplex
inside cells call for small molecules that can modulate these activities
by interacting with G-quadruplexes. However, the paucity of the understanding
of the G-quadruplex stability contributed from submolecular elements,
such as loops and tetraguanine (G) planes (or G-quartets), has hindered
the development of small-molecule binders. Assisted by click chemistry,
herein, we attached pulling handles via two modified guanines in each
of the three G-quartets in human telomeric G-quadruplex. Mechanical
unfolding using these handles revealed that the loop interaction contributed
more to the G-quadruplex stability than the stacking of G-quartets.
This result was further confirmed by the binding of stacking ligands,
such as telomestatin derivatives, which led to similar mechanical
stability for all three G-quartets by significant reduction of loop
interactions for the top and bottom G-quartets. The direct comparison
of loop interaction and G-quartet stacking in G-quadruplex provides
unprecedented insights for the design of more efficient G-quadruplex-interacting
molecules. Compared to traditional experiments, in which mutations
are employed to elucidate the roles of specific residues in a biological
molecule, our submolecular dissection offers a complementary approach
to evaluate individual domains inside a molecule with fewer disturbances
to the native structure