DNA Structure
Capture the diversity of individual DNA molecules and resolve transient structural intermediates
Characterise the DNA conformational landscape with single-molecule tools
The structure of DNA is constantly shifting – through cell cycle phases and interactions with other biomolecules during replication, transcription and repair. Double helices and single-stranded molecules are bent, looped and unwound, initiating key processes and bringing distal parts of the genome together. These processes are highly dynamic and often involve intermediates that may be missed in static or ensemble data.
With in-solution single-molecule Förster resonance energy transfer (smFRET) and Fluorescence correlation spectroscopy (FCS), individual DNA molecules can be tracked in real time with microsecond resolution – ideal for dissecting the folding pathways and topological transitions of DNA structures.
smFRET serves as a sensitive probe for monitoring the proximity of specific nucleotides, enabling the detection of transitions between linear, looped, and multi-stranded geometries or resolving the relative orientations of helical arms in branched DNA/RNA architectures (e.g., Holliday junctions or ribozymes). FCS can differentiate between diffusion coefficients of individual DNAs and DNA complexes, enabling quantification of strand hybridisation or helicase binding.
Visualise dynamic DNA structures with single-molecule clarity
Observe higher-order structures and bring DNA structures to life by augmenting your structural biology toolkit.
- Explore DNA structures in solution, complementing existing biochemical and biophysical data
- Quantify dynamic nanoscale distances between DNA bases with Å precision
- Investigate the impact of environmental factors, such as pH and salt concentration, on DNA structures
- Quantify rapid conformational changes and identify dynamic intermediates that are invisible in ensemble methods
- Observe folding or recombination intermediates, such as branch migration and junction dynamics
- Perform stoichiometric analysis of multi-strand or protein-DNA assemblies
- Observe 'breathing' dynamics or transient base-pair fraying under physiological conditions
smFRET can detect the dynamic interconversion of DNA hairpin states
DNA hairpins can form both open and closed states, making them ideal models for smFRET analysis. Here we show how smFRET can detect both open and closed conformations, and how burst variance analysis indicates that these states are rapidly interconverting. The influence of environmental factors, such as salt concentration, on the rate of interconversion can also be investigated.
Right: FRET efficiency histogram and burst variance analysis of DNA hairpin at 400 mM NaCl; FRET efficiency histograms (top), burst variance analysis (bottom)
Data generated in-house at Exciting Instruments.
DNA structure papers and pre-prints featuring EI-FLEX data
DNA structure FAQs
How does smFRET monitor the opening and closing of a DNA hairpin?
By placing a FRET pair at the base of the stem, its stability can be tracked. High FRET efficiency indicates that the stem is zipped (hairpin closed), while low FRET efficiency indicates the stem has opened. This allows you to measure the conformational changes of the hairpin in real-time in response to environmental changes, such as salt concentration.
Can I use smFRET to study Holliday junctions?
How does FCS detect the melting of a DNA duplex?
Double-stranded DNA (dsDNA) diffuses differently compared to when it has separated into two single-stranded DNA (ssDNA) molecules. By monitoring the diffusion time as temperature changes or denaturants are added, FCS can accurately determine DNA melting profiles.
Can smFRET resolve DNA breathing or denaturation bubbles?
smFRET is ideal for detecting these microsecond-scale fluctuations when dye pairs are placed close to the site of interest; FRET histograms will broaden beyond the expected distribution, and burst variance analysis can detect the dynamic movements of DNA breathing. The impact of factors such as temperature, sequence composition, and supercoiling on the longevity of denaturation bubbles can also be measured.
How can I study the hybridisation of two DNA strands?
By labelling each strand a different colour, FCCS can directly capture duplex formation in solution; a cross-correlation signal will only be generated upon hybridisation. FCCS is more precise than FCS in this context because the mass change is likely to be small.
Can smFRET track branch migration in DNA?
During DNA repair or nanotechnology processes, a DNA branch can slide along the sequence. By placing a donor at a fixed point and an acceptor on the moving strand, you can watch the FRET signal change gradually as the branch moves closer to or further from the reference dye.
How do intercalating dyes or drugs affect DNA structure?
Intercalation of drugs between bases stiffens and lengthens DNA. In FCS, this induces a change in diffusion time; in smFRET, the distance between dyes will increase, leading to a measurable drop in FRET efficiency.
Can I use these techniques to detect the effects of epigenetic marks on DNA accessibility?
FCS can capture how epigenetic marks change the recruitment rate of transcription factors. smFRET can provide information on how methylation and other markers change genomic structure, from DNA accessibility and interactions to remodelling.
Can smFRET detect structural changes at a DNA nick during repair?
Yes – the binding of repair proteins onto nicked DNA can induce a dramatic structural shift. By placing FRET dyes on either side of the nick, smFRET can detect the transition from a straight DNA duplex to a highly kinked conformation, including intermediate states.
How can these techniques investigate DNA supercoiling?
smFRET can detect torsional stress, such as during the formation of plectonemes, or how this exertion results in secondary structures. FCS can measure how compact a DNA molecule is, providing information on the formation or relaxation of supercoiled structures. These techniques are highly complementary with magnetic tweezers that can induce a specific amount of supercoiling.
“What sets the EI-FLEX apart is its versatility - it's our go-to for probing kinetics and conformational changes that the other systems can't resolve. This opens up a range of research for us that was previously impossible.”
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