A nanoplasmonic molecular ruler for measuring nuclease activity and DNA footprinting

Interactions between nucleic acids and proteins are essential to genetic information processing. The detection of size changes in nucleic acids is the key to mapping such interactions, and usually requires substrates with fluorescent, electrochemical or radioactive labels1–3. Recently, methods have been developed to tether DNA to highly water-soluble Au nanoparticles4–8, and nanoparticle pairs linked by DNA have been used to measure nanoscale distances9. Here we demonstrate a molecular ruler in which double-stranded DNA is attached to a Au nanoparticle. The change in plasmon resonance wavelength of individual Au–DNA conjugates depends on the length of the DNA and can be measured with subnanometre axial resolution. An average wavelength shift of approximately 1.24 nm is observed per DNA base pair. This system allows for a label-free, quantitative, real-time measurement of nuclease activity and also serves as a new DNA footprinting platform, which can accurately detect and map the specific binding of a protein to DNA.

Design of the Au–DNA nanoplasmonic molecular ruler. a, Synthesis process of the Au–DNA nanoconjugate. The 20-nm Au nanoparticle modified with a phosphine surfactant monolayer was enclosed by a layer of synthesized 54-bp dsDNA. A thiol group and the FITC (fluorescein isothiocyanate) fluorophore (as indicated by green star) were synthesized at each end of the dsDNA, respectively. Through the thiol–Au chemistry, the dsDNA was tethered onto the Au nanoparticles. b, The dsDNA contains endonuclease incision sites positioned at 12, 24, 36 and 48 bp from the Au-nanoparticle-tethered end. The fluorescent labelling (FITC) is only for further confirmation of the nuclease reactions, and is not necessary for plasmon resonance measurements.

DNA footprinting of Bal31 exonuclease stalled by the EcoRI(Q111) proteins. a, Schematic diagram of footprinting by Bal31 and the stalled Bal31 hydrolysis by DNA-bound EcoRI(Q111) protein. The EcoRI(Q111) blocks nucleotide removal by Bal31 on a single Au–DNA nanoconjugate. b,c, Time-lapse scattering spectra of the single Au–DNA nanoconjugates without EcoRI(Q111) (b) and with EcoRI(Q111) (c) during exonuclease Bal31 hydrolysis. d, Plasmon resonance wavelength of the Au–DNA nanoconjugate as a function of time in the exonuclease reactions, with wavelength shift shown on the right vertical axis and the base pair of the dsDNA removed on the left vertical axis. The error bars represent the standard deviation in the measurement of 10 nanoparticles.