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.
Highlights
Deoxyribonucleic acid (DNA) hybridisation, especially of short DNAs, is an essential process in biology, much is still unknown about the exact process of hybridisation and its kinetics
• DNA hybridisation is a key for biological functions
more informed picture of the kinetics of DNA hybridisation will allow for greater advancements
Summary
Deoxyribonucleic acid (DNA) hybridisation, especially of short DNAs, is an essential process in biology, much is still unknown about the exact process of hybridisation and its kinetics. Single-molecule experiments show terminal labelling with a cyanine fluorescent dye (Cy3) stabilises hybridisation by stacking with terminal bases, reducing koff , and leads to an increase in kon of oligonucleotides [33,44,45]. At the single-molecule level, where DNA hybridisation kinetics can be directly imaged, the DNA sequence used for multiple binding sites has been seen to affect the kon of individual probes. The maximum concentration of labelled ssDNAs in single-molecule TIRF microscopy measurements is limited to 50–100 nM, due to the fluorescence background produced by unbound DNA which if too high can prevent the detection of single molecules Another external factor during DNA hybridisation is the temperature, and as transient hybridisation is thermally driven, it is obvious that the temperature of the measurement will affect the kinetics DNA hybridisation. Kon has been seen to slightly decrease with increasing temperature [3], possibly due to increased events where the ssDNA dissociates whilst in the process of trying to hybridise – known as abortive hybridisation
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