Ensemble Force Spectroscopy of a G-Quadruplex Cluster on a Single-Molecule Platform.

Single-molecule methods offer high sensitivities with precisions superior to bulk assays. However, these methods are low in throughput and cannot repetitively interrogate the same cluster of molecular units. In this work, we investigate a tandem array of G-quadruplexes on a single-molecule DNA template with a throughput of at least two orders of magnitude higher than single-molecule force spectroscopy.

A translation control module coordinates germline stem cell differentiation with ribosome biogenesis during Drosophila oogenesis.

Ribosomal defects perturb stem cell differentiation, and this is the cause of ribosomopathies. How ribosome levels control stem cell differentiation is not fully known. Here, we discover that three DExD/H-box proteins govern ribosome biogenesis (RiBi) and Drosophila oogenesis.

Ensemble Sensing Using Single-Molecule DNA Copolymers.

While single-molecule sensing has offered ultimate mass sensitivity at the precision of individual molecules, it requires a longer time to detect analytes at lower concentrations when analyte binding to single-molecule probes becomes diffusion-limited. Here, we solved this accuracy problem in the concentration sensitivity determination by using single-molecule DNA homopolymers, in which up to 473 identical sensing elements (DNA hairpins) were introduced by rolling circle amplification. Surprisingly, the DNA homopolymers containing as few as 10 tandem hairpins displayed ensemble unfolding/refolding transitions, which were exploited to recognize microRNAs (miRNAs) that populated unfolded hairpins.

Fatal attraction: The roles of ribosomal proteins in the viral life cycle.

Upon viral infection of a host cell, each virus starts a program to generate many progeny viruses. Although viruses interact with the host cell in numerous ways, one critical step in the virus life cycle is the expression of viral proteins, which are synthesized by the host ribosomes in conjunction with host translation factors. Here we review different mechanisms viruses have evolved to effectively seize host cell ribosomes, the roles of specific ribosomal proteins and their posttranslational modifications on viral RNA translation, or the cellular response to infection.

Mechanical Cooperativity in DNA Cruciform Structures.

Unlike short-range chemical bonds that maintain chemical properties of a biological molecule, long-range mechanical interactions determine mechanochemical properties of molecules. Limited by experimental approaches, however, direct quantification of such mechanical interactions is challenging. Using magneto-optical tweezers, herein we found torque can change the topology and mechanochemical property of DNA cruciform, a naturally occurring structure consisting of two opposing hairpin arms.

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.

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

Sensitivity of biosensors is set by the dissociation constant (K) 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 K, they compromise temporal resolution. Here, we demonstrated mechanochemical sensing that broke the K limit by 9 orders of magnitude for Hg detection without amplifications.

Exploded view of higher order G-quadruplex structures through click-chemistry assisted single-molecule mechanical unfolding.

Due to the long-range nature of high-order interactions between distal components in a biomolecule, transition dynamics of tertiary structures is often too complex to profile using conventional methods. Inspired by the exploded view in mechanical drawing, here, we used laser tweezers to mechanically dissect high-order DNA structures into two constituting G-quadruplexes in the promoter of the human telomerase reverse transcriptase (hTERT) gene. Assisted with click-chemistry coupling, we sandwiched one G-quadruplex with two dsDNA handles while leaving the other unit free.

A molecular tuning fork in single-molecule mechanochemical sensing.

The separate arrangement of target recognition and signal transduction in conventional biosensors often compromises the real-time response and can introduce additional noise. To address these issues, we combined analyte recognition and signal reporting by mechanochemical coupling in a single-molecule DNA template. We incorporated a DNA hairpin as a mechanophore in the template, which, under a specific force, undergoes stochastic transitions between folded and unfolded hairpin structures (mechanoescence).

Dissecting cooperative communications in a protein with a high-throughput single-molecule scalpel.

Miscued communication often leads to misfolding and aggregation of the proteins involved in many diseases. Owing to the ensemble average property of conventional techniques, detailed communication diagrams are difficult to obtain. Mechanical unfolding affords an unprecedented perspective on cooperative transitions by observing a protein along a trajectory defined by two mutated cysteine residues.

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).

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.

Fluorescent nanonetworks: a novel bioalley for collagen scaffolds and tissue engineering.

Native collagen is arranged in bundles of aligned fibrils to withstand in vivo mechanical loads. Reproducing such a process under in vitro conditions has not met with major success. Our approach has been to induce nanolinks, during the self-assembly process, leading to delayed rather than inhibited fibrillogenesis.

Influence of functionalized nanoparticles on conformational stability of type I collagen for possible biomedical applications.

Collagen-nanoparticle interactions are vital for many biomedical applications including drug delivery and tissue engineering applications. Iron oxide nanoparticles synthesized using starch template according to our earlier reported procedures were functionalized by treating them with Gum Arabic (GA), a biocompatible polysaccharide, so as to enhance the interaction between nanoparticle surfaces and collagen. Viscosity, circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) techniques have been used to study the collagen-nanoparticle interactions.

Enhancing collagen stability through nanostructures containing chromium(III) oxide.

Stabilization of collagen for various applications employs chemicals such as aldehydes, metal ions, polyphenols, etc. Stability against enzymatic, thermal and mechanical degradation is required for a range of biomedical applications. The premise of this research is to explore the use of nanoparticles with suitable functionalization/encapsulation to crosslink with collagen, such that the three dimensional architecture had the desired stability.

Structurally modified 1,10-phenanthroline based fluorophores for specific sensing of Ni2+ and Cu2+ ions.

We report two fluorophores with open coordination sites for specific sensing of Ni(2+) and Cu(2+) ions via a simple synthetic route. The fluorescence activity was completely quenched on coordination of the metal ions with the phenanthroline ring present in the fluorophore as is clearly evident from the photophysical studies.