nanomanipulation-of-single-rna-molecules-by-optical-tweezers

Nanomanipulation of Single RNA Molecules by Optical Tweezers

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be ...

University at Albany, State University of New York

To the authors' knowledge, no satisfactory therapy is available for patients with refractory germ cell neoplasms. The activity and safety of arsenic trioxide in refractory germ cell tumors was assessed.

Southwest Oncology Group phase II study of arsenic trioxide in patients with refractory germ cell malignancies.

The pathology of sickle cell disease arises from the occlusion of small blood vessels because of polymerization of the sickle hemoglobin within the red cells. We present measurements using a microfluidic method we have developed to determine the pressure required to eject individual red cells from a capillary-sized channel after the cell has sickled. We find that the maximum pressure is only ∼100 Pa, much smaller than typically found in the microcirculation. This explains why experiments using animal models have not observed occlusion beginning in capillaries. The magnitude of the pressure and its dependence on intracellular concentration are both well described as consequences of sickle hemoglobin polymerization acting as a Brownian ratchet. Given the recently determined stiffness of sickle hemoglobin gels, the observed obstruction seen in sickle cell disease as mediated by adherent cells can now be rationalized, and surprisingly suggests a window of maximum vulnerability during circulation of sickle cells.

The physical foundation of vasoocclusion in sickle cell disease.

Fascicular tachycardia (FT) is an uncommon cause of monomorphic sustained ventricular tachycardia (VT). We describe 6 cases of FT with multiform QRS morphologies.

Diagnosis and ablation of multiform fascicular tachycardia.

In minimal RNA kissing complexes formed between hairpins with cognate GACG tetraloops, the two tertiary GC pairs are likely stabilized by the stacking of 5'-unpaired adenines at each end of the short helix. To test this hypothesis, we mutated the flanking adenines to various nucleosides and examined their effects on the kissing interaction. Electrospray ionization mass spectrometry was used to detect kissing dimers in a multiequilibria mixture, whereas optical tweezers were applied to monitor the (un)folding trajectories of single RNA molecules. The experimental findings were rationalized by molecular dynamics simulations. Together, the results showed that the stacked adenines are indispensable for the tertiary interaction. By shielding the tertiary base pairs from solvent and reducing their fraying, the stacked adenines made terminal pairs act more like interior base pairs. The purine double-ring of adenine was essential for effective stacking, whereas additional functional groups modulated the stabilizing effects through varying hydrophobic and electrostatic forces. Furthermore, formation of the kissing complex was dominated by base pairing, whereas its dissociation was significantly influenced by the flanking bases. Together, these findings indicate that unpaired flanking nucleotides play essential roles in the formation of otherwise unstable two-base-pair RNA tertiary interactions.

The essential role of stacking adenines in a two-base-pair RNA kissing complex.

RNA folding in cells typically occurs at mesophilic temperatures. However, in vitro, RNA can be unfolded either by increasing temperature to values that are much higher than physiological, or by mechanically pulling structures apart at ambient temperature. To directly study RNA folding at physiological temperatures and to unify thermodynamics measured by melting and pulling, we developed temperature-controlled optical tweezers (thermal tweezers) that can be used to mechanically unfold single RNA molecules at mesophilic temperatures. Folding of a 20-base-pair tetraloop hairpin was studied under different ionic conditions and at temperatures ranging from 22 °C to 42 °C. At each temperature, single hairpin molecules were held at constant force, and their two-state folding equilibria were monitored. The change in free energy derived from these measurements was used to construct a phase diagram of RNA structure using force and temperature as variables. Furthermore, we derived ΔG(0pN,T), the folding free energy at zero force and temperature T, by subtracting the stretching energy of unfolded RNA from the reversible mechanical work done to unfold the hairpin. ΔG(0pN,T) and its salt dependence agree reasonably well with the predictions by the nearest neighbor model. Under each ionic condition, ΔG(0pN,T) depended linearly on temperature, yielding ΔH(exp) and ΔS(exp) that also matched the predictions. The combination of force and temperature to study RNA folding is a step toward unifying thermodynamics measured by thermal melting and mechanical unfolding, and opens a new path for directly monitoring temperature induced RNA structural changes, as it occurs often in biology.

William Stephenson

Nanoscale Engineering Graduate Program,
College of Nanoscale Science and Engineering,
Nanoscale Engineering Graduate Program, College of Nanoscale Science and Engineering

University at Albany, State University of New York

Nanomanipulation of Single RNA Molecules by Optical Tweezers

William Stephenson

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Nanoscale Engineering Graduate Program,College of Nanoscale Science and Engineering,Nanoscale Engineering Graduate Program, College of Nanoscale Science and Engineering

University at Albany, State University of New York

Nanomanipulation of Single RNA Molecules by Optical Tweezers

Nanoscale Engineering Graduate Program,
College of Nanoscale Science and Engineering,
Nanoscale Engineering Graduate Program, College of Nanoscale Science and Engineering