Observation of Ethanol-Induced Condensation and Decondensation Processes at a Single-DNA Molecular Level in Microfluidic Devices Equipped with a Rapid Solution Exchange System

Abstract

Conformational transitions from secondary (e.g., B-to A-form DNA) to higher-order (e.g., coil to globule) transitions play important roles in genome expression and maintenance. Several singlemolecule approaches using microfluidic devices have been used to determine the kinetics of DNA chromatin assembly because microfluidic devices can afford stretched DNA molecules through laminar flow and rapid solution exchange. However, some issues, particularly the uncertainty of time 0 in the solution exchange process, are encountered. In such kinetic experiments, it is critical to determine when the target solution front approaches the target DNA molecules. Therefore, a new design for a microfluidic device is developed that enables the instantaneous exchange of solutions in the observation channel, allowing accurate measurements of DNA conformational transitions; stepwise, ethanol-induced conformational transitions are revealed. Although full DNA contraction from coil to globule is observed with >50% ethanol, no outstanding change is observed at concentrations <40% in 10 min. With 50% ethanol solution, the DNA conformational transition passes through two steps: (i) fast and constant-velocity contraction and (ii) relatively slow contraction from the free end. The first process is attributed to the B to A conformational transition by gradual dehydration. The second process is due to the coil−globule transition as the free end of DNA starts the contraction. This globular structure formation counteracts the shear force from the microfluids and decelerates the contraction velocity. This real-time observation system can be applied to the kinetic analysis of DNA conformational transitions such as kinetics of chromatin assembly and gene expression.

Ethanol-Induced Condensation and Decondensation in DNA-Linked Nanoparticles: A Nucleosome-like Model for the Condensed State

“…Previous theoretical calculations confirm that DNA will adopt B-form conformations in water and A-form in 80% ethanol when it is fully extended, , while single-molecule approaches and CD spectra ,, suggest that the ethanol-induced DNA condensation begins with a B- to A-form DNA transition, followed by a higher-order transition. The structure of this higher-order condensed structure is not known, which is an important need in this field.…”

“…Alternatively, monovalent ion-induced DNA condensation can be achieved even at relatively low ionic concentrations (∼0.3 M NaCl) by adding ethanol, which is a commonly used procedure for precipitating DNA . Circular dichroism (CD) spectra, − electron microscopy, and single-molecule techniques , indicate the existence of a conformational transition from B- to A-form DNA followed by a higher-order transition from extended DNA to toroidal supercoiling in the ethanol-induced condensation of DNA in solution. Inspired by this, Mason, Mirkin, and co-workers designed spherical PAEs and observed an up to 80% decrease in DNA bond length (i.e., the surface-to-surface distance between adjacent nanoparticles that are linked by DNA) shrinks from 16 to 3 nm by adding 80% ethanol, which is much greater than the ∼25% contraction expected for the B- to A-transition in DNA .…”

“…approaches32 and CD spectra27,29,30 suggest that the ethanolinduced DNA condensation begins with a B-to A-form DNA transition, followed by a higher-order transition. The structure of this higher-order condensed structure is not known, which is an important need in this field.…”