Single‐Molecule FRET Ruler Based on Rigid DNA Origami Blocks

Abstract

Fluorescence resonance energy transfer (FRET) has become a work-horse for distance measurements on the nanometer scale and between single molecules. Recent model systems for the FRET distance dependence such as polyprolines and dsDNA suffered from limited persistence lengths and sample heterogeneity. We designed a series of rigid DNA origami blocks where each block is labeled with one donor and one acceptor at distances ranging between 2.5 and 14 nm. Since all dyes are attached in one plane to the top surface of the origami block, static effects of linker lengths cancel out in contrast to commonly used dsDNA. We used single-molecule spectroscopy to compare the origami-based ruler to dsDNA and found that the origami blocks directly yield the expected distance dependence of energy transfer since the influence of the linkers on the donor-acceptor distance is significantly reduced. Based on a simple geometric model for the inter-dye distances on the origami block, the Förster radius R(0) could directly be determined from the distance dependence of energy transfer yielding R(0)=5.3±0.3 nm for the Cy3-Cy5 pair.

Distance Dependence of Single-Fluorophore Quenching by Gold Nanoparticles Studied on DNA Origami

“…6À10 The DNA origami technique additionally offers the possibility to place various moieties such as fluorescent dyes on a rigid DNA scaffold with subnanometer accuracy. 11,12 Here, we have combined these unique abilities and revisited the distance-dependent interaction of a single fluorescent dye with a single gold nanoparticle.The interaction between metallic nanoparticles and fluorophores has recently gained considerable attention. 13,14 On one hand, the fluorescent dye is a nanoreporter of its immediate dielectric environment.…”

Random coil negative control reproduces the discrepancy between scattering and FRET measurements of denatured protein dimensions

“…Similarly, although smFRET, by comparison, relies on several denaturantdependent assumptions and approximations, no attempt to explain the discrepancy between smFRET and scattering as arising because of these assumptions and approximations has yet proven successful (4). Studies of rigid constructs, such as polyproline (23,(28)(29)(30)(31)(32), for example, indicate that the denaturant dependence of the index of refraction, the quantum yield, and the spectral shift of the dyes are all too small to account for the observed changes in transfer efficiency. Denaturant-dependent viscosity effects on the rate of conformational averaging have,…”

FRET efficiency and antenna effect in multi-color DNA origami-based light harvesting systems

“…21,22 Since every staple strand can be addressed and modied individually and separately, different moieties can be arranged with a high local control since the exact position of each staple strand in the DNA origami structure is known. DNA origami structures have been used to create highly sensitive SERS substrates by attaching gold nanoparticle dimers, [23][24][25] to analyze DNA strand breaks induced by low energy electrons 26,27 and UV photons 28 and to arrange different uo-rophores 29,29,30 at precise distances to create nanoscale photonic devices which can be used for example as photonic wires, 15,18 to resolve conformational changes of biomolecules, [31][32][33][34] as logic gates 35,36 and articial light harvesting complexes. 8,10,18 The light harvesting efficiency is in this context typically expressed as an antenna effect (AE), i.e.…”