Force-measuring Optical Tweezers Research Articles

Effect of Methylation on the Nanomechanics of Double-Stranded DNA

In its physiological environment DNA is constantly exposed to mechanical stress. The nanomechanical properties of DNA influence not only its response to stress but also its interaction with proteins. Despite its crucial role in epigenetics, little is known about how methylation affects the nanomechanical properties of DNA. To investigate the impact of methylation on DNA nanomechanics, here we manipulated single molecules of chemically or enzymatically methylated DNA and compared their properties with those of non-methylated DNA. As a model we used a 3312-base-pair long sequence of lambda-phage DNA that met the criteria of a CpG island. Chemically methylated DNA was prepared with PCR containing 5-methyl-CTP in the reaction mixture. For enzymatic methylation the M.Sss.I methyltransferase was used. Single DNA molecules were mechanically manipulated with force-measuring optical tweezers in repeated stretch-relaxation cycles. Surface-adsorbed DNA molecules were studied by using atomic force microscopy (AFM). We found that the molecular contour length, bending rigidity and intrinsic stiffness were decreased in methylated DNA, pointing at structural and nanomechanical alterations. Furthermore, the cooperative overstretch transition was significantly longer in the methylated form of the molecule, suggesting that the dynamics of intramolecular rearrangements were also affected. AFM measurements of DNA molecules adsorbed to mica surface substantiated the significant reduction of molecular contour length in methylated DNA. By contrast, the apparent bending rigidity of the surface-adsorbed methylated DNA was increased, which is most likely caused by interactions between DNA and the mica surface. In sum, methylation leads to an axial compaction of the dsDNA structure, an increase in bending flexibility in the low-force regime and an increase in axial compliance at higher forces (>20pN). Conceivably, modulation of DNA structure and nanomechanics caused by methylation leads to a complex control of structural accessibility and association kinetics of DNA-binding proteins.

Impairment of the biomechanical compliance of P pili: a novel means of inhibiting uropathogenic bacterial infections?

Gram-negative bacteria often initiate their colonization by use of extended attachment organelles, so called pili. When exposed to force, the rod of helix-like pili has been found to be highly extendable, mainly attributed to uncoiling and recoiling of its quaternary structure. This provides the bacteria with the ability to redistribute an external force among a multitude of pili, which enables them to withstand strong rinsing flows, which, in turn, facilitates adherence and colonization processes critical to virulence. Thus, pili fibers are possible targets for novel antibacterial agents. By use of a substance that compromises compliance of the pili, the ability of bacteria to redistribute external forces can be impaired, so they will no longer be able to resist strong urine flow and thus be removed from the host. It is possible such a substance can serve as an alternative to existing antibiotics in the future or be a part of a multi-drug. In this work we investigated whether it is possible to achieve this by targeting the recoiling process. The test substance was purified PapD. The effect of PapD on the compliance of P pili was assessed at the single organelle level by use of force-measuring optical tweezers. We showed that the recoiling process, and thus the biomechanical compliance, in particular the recoiling process, can be impaired by the presence of PapD. This leads to a new concept in the search for novel drug candidates combating uropathogenic bacterial infections—“coilicides”, targeting the subunits of which the pilus rod is composed.

Investigation of Molecular Level Stress-Strain Relationships in Systems of Entangled F-Actin by Combined Force-Measuring Optical Tweezers and Fluorescence Microscopy

Actin plays a major role in cell structure, cell motility, vesicle and organelle transport, and muscle contraction. Actin's ability to play these major roles is a direct consequence of the intricate relationship between stress and strain in a variety of filamentous actin (F-actin) networks. A thorough understanding of the unique stress-strain relationships in complex F-actin networks at the molecular-level is currently lacking despite the importance of such networks to fields such as biomimetic material engineering and cell biology. Here we develop novel single-molecule instrumentation that combines dual-trap force-measuring optical tweezers with fluorescence microscopy to enable simultaneous characterization of intermolecular forces and molecular dynamics within F-actin networks. This instrumentation is combined with a novel technique in which single F-actin “probes” are used to apply molecular-level strains and measure induced stress within entangled F-actin systems while the deformations and dynamics of surrounding fluorescent-labeled filaments are simultaneously imaged. Specifically, the dual-trap optical tweezers trap fluorescent-labeled, microsphere-conjugated probes and measure the force induced on the probe as it is moved through a network of selectively-labeled entangled F-actin by using a nanoprecison piezoelectric stage to move the sample chamber relative to the traps. Fluorescence microscopy is used simultaneously to image the dynamics of the moving probe as well as the surrounding labeled molecules subject to the applied strain. Specific bioconjugation of the fluorescent polystyrene microspheres to the ends of labeled F-actin is achieved by carbodiimide attachment of gelsolin to microspheres and combining gelsolin-coated microspheres with fluorescent phalloidin labeled actin. This powerful single-molecule technique allows simultaneous measurement of intermolecular forces and dynamics and deformations of single molecules, providing the much needed link between stress and strain at the molecular level in complex F-actin networks.

Fast uncoiling kinetics of F1C pili expressed by uropathogenic Escherichia coli are revealed on a single pilus level using force-measuring optical tweezers

Uropathogenic Escherichia coli (UPEC) express various kinds of organelles, so-called pili or fimbriae, that mediate adhesion to host tissue in the urinary tract through specific receptor-adhesin interactions. The biomechanical properties of these pili have been considered important for the ability of bacteria to withstand shear forces from rinsing urine flows. Force-measuring optical tweezers have been used to characterize individual organelles of F1C type expressed by UPEC bacteria with respect to such properties. Qualitatively, the force-versus-elongation response was found to be similar to that of other types of helix-like pili expressed by UPEC, i.e., type 1, P, and S, with force-induced elongation in three regions, one of which represents the important uncoiling mechanism of the helix-like quaternary structure. Quantitatively, the steady-state uncoiling force was assessed as 26.4 ±1.4 pN, which is similar to those of other pili (which range from 21 pN for S(I) to 30 pN for type 1). The corner velocity for dynamic response (1,400nm/s) was found to be larger than those of the other pili (400-700nm/s for S and P pili, and 6nm/s for type 1). The kinetics were found to be faster, with a thermal opening rate of 17Hz, a few times higher than S and P pili, and three orders of magnitude higher than type 1. These data suggest that F1C pili are, like P and S pili, evolutionarily selected to primarily withstand the conditions expressed in the upper urinary tract.

Unfolding and refolding properties of S pili on extraintestinal pathogenic Escherichia coli

S pili are members of the chaperone-usher-pathway-assembled pili family that are predominantly associated with neonatal meningitis (S(II)) and believed to play a role in ascending urinary tract infections (S(I)). We used force-measuring optical tweezers to characterize the intrinsic biomechanical properties and kinetics of S(II) and S(I) pili. Under steady-state conditions, a sequential unfolding of the layers in the helix-like rod occurred at somewhat different forces, 26 pN for S(II) pili and 21 pN for S(I) pili, and there was an apparent difference in the kinetics, 1.3 and 8.8 Hz. Tests with bacteria defective in a newly recognized sfa gene (sfaX (II)) indicated that absence of the sfaX (II) gene weakens the interactions of the fimbrium slightly and decreases the kinetics. Data of S(I) are compared with those of previously assessed pili primary associated with urinary tract infections, the P and type 1 pili. S pili have weaker layer-to-layer bonds than both P and type 1 pili, 21, 28 and 30 pN, respectively. In addition, the S pili kinetics are ~10 times faster than the kinetics of P pili and ~550 times faster than the kinetics of type 1 pili. Our results also show that the biomechanical properties of pili expressed ectopically from a plasmid in a laboratory strain (HB101) and pili expressed from the chromosome of a clinical isolate (IHE3034) are identical. Moreover, we demonstrate that it is possible to distinguish, by analyzing force-extension data, the different types of pili expressed by an individual cell of a clinical bacterial isolate.

Physical Properties of Biopolymers Assessed by Optical Tweezers: Analysis of Folding and Refolding of Bacterial Pili

Bacterial adhesion to surfaces mediated by specific adhesion organelles that promote infections, as exemplified by the pili of uropathogenic E. coli, is studied mostly at the level of cell-cell interactions and thereby reflects the averaged behavior of multiple pili. The role of pilus rod structure has therefore only been estimated from the outcome of experiments involving large numbers of organelles at the same time. It has, however, lately become clear that the biomechanical behavior of the pilus shafts play an important, albeit hitherto rather unrecognized, role in the adhesion process. For example, it has been observed that shafts from two different strains, even though they are similar in structure, result in large differences in the ability of the bacteria to adhere to their host tissue. However, in order to identify all properties of pilus structures that are of importance in the adhesion process, the biomechanical properties of pili must be assessed at the single-molecule level. Due to the low range of forces of these structures, until recently it was not possible to obtain such information. However, with the development of force-measuring optical tweezers (FMOT) with force resolution in the low piconewton range, it has lately become possible to assess forces mediated by individual pili on single living bacteria in real time. FMOT allows for a more or less detailed mapping of the biomechanical properties of individual pilus shafts, in particular those that are associated with their elongation and contraction under stress. This Mi- nireview presents the FMOT technique, the biological model system, and results from assessment of the biomechanical properties of bacterial pili. The information retrieved is also compared with that obtained by atomic force microscopy.

Interaction of cationic surfactants with DNA: a single-molecule study

The interaction of cationic surfactants with single dsDNA molecules has been studied using force-measuring optical tweezers. For hydrophobic chains of length 12 and greater, pulling experiments show characteristic features (e.g. hysteresis between the pulling and relaxation curves, force-plateau along the force curves), typical of a condensed phase (compaction of a long DNA into a micron-sized particle). Depending on the length of the hydrophobic chain of the surfactant, we observe different mechanical behaviours of the complex (DNA-surfactants), which provide evidence for different binding modes. Taken together, our measurements suggest that short-chain surfactants, which do not induce any condensation, could lie down on the DNA surface and directly interact with the DNA grooves through hydrophobic–hydrophobic interactions. In contrast, long-chain surfactants could have their aliphatic tails pointing away from the DNA surface, which could promote inter-molecular interactions between hydrophobic chains and subsequently favour DNA condensation.

Single-molecule mechanical unfolding and folding of a pseudoknot in human telomerase RNA

RNA unfolding and folding reactions in physiological conditions can be facilitated by mechanical force one molecule at a time. By using force-measuring optical tweezers, we studied the mechanical unfolding and folding of a hairpin-type pseudoknot in human telomerase RNA in a near-physiological solution, and at room temperature. Discrete two-state folding transitions of the pseudoknot are seen at approximately 10 and approximately 5 piconewtons (pN), with ensemble rate constants of approximately 0.1 sec(-1), by stepwise force-drop experiments. Folding studies of the isolated 5'-hairpin construct suggested that the 5'-hairpin within the pseudoknot forms first, followed by formation of the 3'-stem. Stepwise formation of the pseudoknot structure at low forces are in contrast with the one-step unfolding at high forces of approximately 46 pN, at an average rate of approximately 0.05 sec(-1). In the constant-force folding trajectories at approximately 10 pN and approximately 5 pN, transient formation of nonnative structures were observed, which is direct experimental evidence that folding of both the hairpin and pseudoknot takes complex pathways. Possible nonnative structures and folding pathways are discussed.

Direct Observation of the Three-State Folding of a Single Protein Molecule

We used force-measuring optical tweezers to induce complete mechanical unfolding and refolding of individual Escherichia coli ribonuclease H (RNase H) molecules. The protein unfolds in a two-state manner and refolds through an intermediate that correlates with the transient molten globule-like intermediate observed in bulk studies. This intermediate displays unusual mechanical compliance and unfolds at substantially lower forces than the native state. In a narrow range of forces, the molecule hops between the unfolded and intermediate states in real time. Occasionally, hopping was observed to stop as the molecule crossed the folding barrier directly from the intermediate, demonstrating that the intermediate is on-pathway. These studies allow us to map the energy landscape of RNase H.

Mechanism of Force Generation of a Viral DNA Packaging Motor

A large family of multimeric ATPases are involved in such diverse tasks as cell division, chromosome segregation, DNA recombination, strand separation, conjugation, and viral genome packaging. One such system is the Bacillus subtilis phage phi 29 DNA packaging motor, which generates large forces to compact its genome into a small protein capsid. Here we use optical tweezers to study, at the single-molecule level, the mechanism of force generation in this motor. We determine the kinetic parameters of the packaging motor and their dependence on external load to show that DNA translocation does not occur during ATP binding but is likely triggered by phosphate release. We also show that the motor subunits act in a coordinated, successive fashion with high processivity. Finally, we propose a minimal mechanochemical cycle of this DNA-translocating ATPase that rationalizes all of our findings.

Force detection in optical tweezers using backscattered light

In force-measuring optical tweezers applications the position of a trapped bead in the direction perpendicular to the laser beam is usually accurately determined by measuring the deflection of the light transmitted through the bead. In this paper we demonstrate that this position and thus the force exerted on the bead can be determined using the backscattered light. Measuring the deflection for a 2.50 mum polystyrene bead with both a position sensitive detector (PSD) and a quadrant detector (QD) we found that the linear detection range for the PSD is approximately twice that for the QD. In a transmission-based setup no difference was found between both detector types. Using a PSD in both setups the linear detection range for 2.50 mum beads was found to be approximately 0.50 mum in both cases. Finally, for the reflection-based setup, parameters such as deflection sensitivity and linear detection range were considered as a function of bead diameter (in the range of 0.5-2.5 mum). 140pN was the largest force obtained using 2.50 mum beads.