DNA hybridisation kinetics using single-molecule fluorescence imaging

Abstract

Deoxyribonucleic acid (DNA) hybridisation plays a key role in many biological processes and nucleic acid biotechnologies, yet surprisingly there are many aspects about the process which are still unknown. Prior to the invention of single-molecule microscopy, DNA hybridisation experiments were conducted at the ensemble level, and thus it was impossible to directly observe individual hybridisation events and understand fully the kinetics of DNA hybridisation. In this mini-review, recent single-molecule fluorescence-based studies of DNA hybridisation are discussed, particularly for short nucleic acids, to gain more insight into the kinetics of DNA hybridisation. As well as looking at single-molecule studies of intrinsic and extrinsic factors affecting DNA hybridisation kinetics, the influence of the methods used to detect hybridisation of single DNAs is considered. Understanding the kinetics of DNA hybridisation not only gives insight into an important biological process but also allows for further advancements in the growing field of nucleic acid biotechnology.

Can DyeCycling break the photobleaching limit in single-molecule FRET?

“…A wealth of information is available on hybridization kinetics [37,41,43,46]. The hybridization rate depends on the cycler's diffusion time, and is tunable with cycler concentration, temperature, and salt concentration, whereas the dissociation rate can be tuned via the DNA duplex length and sequence (GC content, mismatches [41,47]), salt conditions, and temperature [41]. In addition, synthetic nucleic acid analogues have been developed to overcome potential shortcomings of DNA [48], such as its negative charge, relatively low affinity, and susceptibility to degradation.…”

Free-energy landscapes of DNA and its assemblies: Perspectives from coarse-grained modelling

“…Hybridization, the association of two strands to form a double helix, represents the most fundamental process of DNA self-assembly. Consequently, this process has been well characterized experimentally with the thermodynamics well understood [5,15] but with open questions still remaining concerning the hybridization kinetics [16,17].…”

Comparing Transient Oligonucleotide Hybridization Kinetics Using DNA-PAINT and Optoplasmonic Single-Molecule Sensing on Gold Nanorods

“…Once a docking strand is bound, the probability that dissociation does not take place in an interval 𝑡 𝑏 (bound waiting time) is 𝑃(𝑡 𝑏 ) ≃ exp(−𝑘 off 𝑡 𝑏 ), where 𝑘 off is the dissociation rate constant and, in this case, equivalent to a single molecule dissociation rate with units of 𝑠 −1 . In other words, we expect 𝑘 off to be concentration-independent 28,29 . Note that the approximation holds provided that the chance of two imagers being bound at the same time is negligible, which is achieved by making the imager concentration sufficiently low.…”

“…The laser sweep frequency is 50Hz, and the tracking of the resonance position occurs at the 20 ms time intervals. This experiment is also conducted at 22.5 o C.DNA hybridization has previously been reported28,29 to follow (pseudo) first-order kinetics. Considering a single docking strand that can be found in two states, either bounded with a single imager strand or dissociated, the probability that a binding event does not take place in an interval 𝑡 𝑑 (dissociated waiting time), also called survival probability, is 𝑃 𝑠 (𝑡 𝑑 ) = exp (−𝑘 𝑠 𝑡 𝑑 ) where 𝑘 𝑠 is a singlemolecule binding rate with units of 𝑠 −1 (also known as association rate).…”