A Synthetic DNA Walker for Molecular Transport

Abstract

Materials and methodsOligonucleotide and Device Preparation. All DNA oligonucleotides were synthesized by Integrated DNA Technologies, Inc. DNA labelling with fluorophore or quencher and subsequent purification were performed by the supplier and the resulting oligomers were used without further purification. DNA stock solutions (40-60 µM) were prepared in TE buffer (pH 8.0) and concentrations were determined at 260 nm using the molecular extinction coefficient supplied by the manufacturer. The track (final concentration = 5 µM) was prepared by mixing the six track strand species in TSE buffer (10 mM Tris, 150 mM NaCl, 1 mM EDTA, pH 7.5), followed by incubation for 3 hr at 37• C. The walker was prepared by the same procedure using the two walker strand species.Non-denaturing PAGE Analysis. Fuel-mediated association of the walker and track was analyzed by non-denaturing PAGE. The reaction volume for the PAGE experiments was 10 µl. Initial anchoring of the walker on branch 1 was achieved by adding equimolar A1 to a reaction mixture of track and walker (1 µM in TSE buffer), followed by 1 hr incubation at 37• C. Subsequent walker movement was carried out by successively adding different fuel species in equimolar amounts. Each addition was followed by 1 hr incubation at 37• C. The whole reaction sample was loaded in a 6.7% non-denaturing gel. Gel electrophoresis was accomplished in 1× TBE buffer at 50 V and 4• C, and the bands were visualized by fluorescent scanning (Molecular Imager FX Pro Plus, Bio-Rad).Multiplexed Real-time Fluorescence Measurement. Fluorescence measurements were carried out with a fluorometer (PTI Co.) at room temperature. Bandwidths for excitation and emission were set to 4 nm and the working volume for measurements was 100 µl. Track (0.5 µM) was preincubated with equimolar walker and A1 in TSE buffer for 4 hr at room temperature. Equimolar amounts of fuel strands were successively added from 100× stocks and the solution was mixed by rapid pipetting. Fluorescence signals from four different dyes were collected during the same run using the multi-dye mode (Felix32 software, PTI Co.) to monitor fluorescence intensities at four excitation/emission wavelengths. Independence of fluorescence signalsTo monitor walker movement, four fluorescent dyes were selected (FAM, HEX, Texas Red, Cy5 in order of increasing excitation/emission wavelengths) from many possible candidates. To minimize FRET between dyes, branches 1-4 on the track were labeled in the order (HEX, Cy5, FAM, Texas Red) to double the distance between dyes with adjacent fluorescence spectra. These concerns were particularly relevant because the equilibrium distance between neighboring dyes (5 nm) is typical of the Förster radius for many dye pairs. Excitation and emission wavelengths for each dye were chosen so as to minimize the response of the other three dyes. Columns 2-5 of Table S1 list fluorescence intensities of the four dye-labled T strands at the four excitation/emission wavelengths employed for multiplexed fluorescence measurements. ...

Programmable disorder in random DNA tilings

“…The first generation of DNA robots were developed to make a few steps on one-dimensional tracks consisting of a double helix. [21][22][23] After DNA origami was invented, it was used as a two-dimensional playground for DNA robots with more interesting functions, including following a path, 24 picking up cargos in an assembly line, 25 and making choices at multiple junctions. 26 The complexity of playgrounds for DNA robots not only affects the functions that can be demonstrated, but also determines how we can evaluate the designed behaviors.…”

Direct observation of stepwise movement of a synthetic molecular transporter

“…This precisely controlled, long-range transport could lead to the development of systems that could be programmed and routed by instructions encoded in the nucleotide sequences of the track and motor. Such systems might be used to create molecular assembly lines modelled on the ribosome.An effective linear molecular motor must traverse its track without dissociating [1][2][3][4][5][6][7]10,12 and run unidirectionally without external intervention [4][5][6][7][8][9][10][11][12] . Directionality may be imposed by the sequential addition of DNA instructions 1-3 or, for autonomous motors, by modifying the track sites that have been visited 5,6,12 , by coupling motion to a unidirectional reaction cycle 4,9,12 or by coordinating the conformation changes of different parts of the motor 11,12 .…”

“…Controlled motion at the nanoscale can be achieved by using Watson-Crick base-pairing to direct the assembly and operation of a molecular transport system consisting of a track, a motor [1][2][3][4][5][6][7][8][9][10][11][12] and fuel [13][14][15] , all made from DNA. Here, we assemble a 100-nm-long DNA track on a two-dimensional scaffold 16 , and show that a DNA motor loaded at one end of the track moves autonomously and at a constant average speed along the full length of the track, a journey comprising 16 consecutive steps for the motor.…”

“…Directionality may be imposed by the sequential addition of DNA instructions [1][2][3] or, for autonomous motors, by modifying the track sites that have been visited 5,6,12 , by coupling motion to a unidirectional reaction cycle 4,9,12 or by coordinating the conformation changes of different parts of the motor 11,12 . DNA motors that satisfy all these criteria have typically been demonstrated on tracks that allow only 1-3 steps, although a stochastic DNA 'spider' with many legs has been shown to move longer distances by biased diffusion 17 along a 100 nm track 18 .…”

Scaffolded DNA Origami: from Generalized Multicrossovers to Polygonal Networks

“…At an intellectual level the technical achievements of the resulting collaborations and interactions have been significant, among them the first two-dimensional DNA crystals [4] and algorithmic self-assembly of both linear [5] and two-dimensional [6] arrays. By various other paths, a number of physicists have joined the party, mixing their own ideas with Ned's paradigm of "DNA as tinkertoys" to create nanomechanical systems such as DNA tweezers [7] and walkers [8,9,10]. DNA nanotechnology has taken on a life of its own since Ned's original vision of DNA fish flying in an extended Escherian lattice [11] and we look forward to a new "DNA world" in which an all-DNA "bacterium" wriggles, reproduces and computes.…”