Single-Molecule Mechanics in Ligand Concentration Gradient

Abstract

Single-molecule experiments provide unique insights into the mechanisms of biomolecular phenomena. However, because varying the concentration of a solute usually requires the exchange of the entire solution around the molecule, ligand-concentration-dependent measurements on the same molecule pose a challenge. In the present work we exploited the fact that a diffusion-dependent concentration gradient arises in a laminar-flow microfluidic device, which may be utilized for controlling the concentration of the ligand that the mechanically manipulated single molecule is exposed to. We tested this experimental approach by exposing a λ-phage dsDNA molecule, held with a double-trap optical tweezers instrument, to diffusionally-controlled concentrations of SYTOX Orange (SxO) and tetrakis(4-N-methyl)pyridyl-porphyrin (TMPYP). We demonstrate that the experimental design allows access to transient-kinetic, equilibrium and ligand-concentration-dependent mechanical experiments on the very same single molecule.

TMPyP binding evokes a complex, tunable nanomechanical response in DNA

“…The length increment could be completely inhibited by 1 M NaCl, indicating that the mechanism of TMPyP binding to DNA is electrostatic. It has been shown before that TMPyP intercalates into DNA (66)(67)(68)(69), and that intercalators cause DNA lengthening (6,8,10,12,13,(48)(49)(50)(51). Thus, we conclude that the primary length increment is caused by TMPyP intercalation.…”

“…To obtain the force curve of a single DNA molecule, first it was pulled in buffer without TMPyP (control measurement) at a constant, pre-adjusted pulling rate. Subsequently, the molecule was brought to the microfluidic channel containing TMPyP at the given concentration, and it was positioned far from the channel wall so that diffusion-driven transport processes would not influence the effective TMPyP concentration (6,8). Then, the pulling cycle was repeated with the same rate.…”

“…DNA has particularly been in the focus of single-molecule experiments, as the molecule lends itself naturally to exploring the role of its axial, bending, twisting and strand-separation nanomechanics in DNA-associated processes (1)(2)(3)(4)(5). The combination of novel methodologies, for example, optical tweezers, microfluidics and high-resolution fluorescence microscopies, has paved the way towards understanding how DNA nanomechanics are influenced by external factors such as intercalators (6), binding proteins (7) or solute concentration gradients (8). Understanding DNA nanomechanics is key not only to uncover the mechanisms of the vast array of DNA-related intracellular processes, for instance, DNA replication and repair, chromatin condensation, gene transcription and regulation, but also in the development of novel, high-efficacy pharmaceuticals that specifically and sensitively influence these DNAdependent processes (9,10).…”

Editorial for the Special Issue on Optical Trapping and Manipulation: From Fundamentals to Applications

“…Optical tweezers also find application in single-molecule biophysics, such as the work reported by Kretzer et al [4] By combining optical tweezers into a microfluidic device, the authors were able to measure the concentration-dependent effects on the mechanics of single molecule ligand binding.…”