Switchable DNA interfaces for the highly sensitive detection of label-free DNA targets

Abstract

We report a method to detect label-free oligonucleotide targets. The conformation of surface-tethered probe nucleic acids is modulated by alternating electric fields, which cause the molecules to extend away from or fold onto the biased surface. Binding (hybridization) of targets to the single-stranded probes results in a pronounced enhancement of the layer-height modulation amplitude, monitored optically in real time. The method features an exceptional detection limit of <3 ؋ 10 8 bound targets per cm 2 sensor area. Single base-pair mismatches in the sequences of DNA complements may readily be identified; moreover, binding kinetics and binding affinities can be determined with high accuracy. When driving the DNA to oscillate at frequencies in the kHz regime, distinct switching kinetics are revealed for single-and double-stranded DNA. Molecular dynamics are used to identify the binding state of molecules according to their characteristic kinetic fingerprints by using a chip-compatible detection format.fluorescence ͉ sensor ͉ SNP ͉ affinity ͉ energy transfer

Real-time kinetics measurements of protein induced conformational changes in DNA

“…The switching efficiency corresponds to the difference in the fluorescent intensity between DNA in lying (positive applied potential) and standing (negative applied potential) orientation. This is highly correlated to surface layer density [4]. As the DNA layer is desorbed the switching efficiency initially increases due to reduced steric hindrance between adjacent DNA probes until it peaks and drops due to an overall decrease in fluorescent intensity against a constant background (see [8] for a detailed description).…”

“…This allows for real time observation of the emitter -gold distance through measurement of the fluorescent intensity [2]. As the contour length of the tethered dsDNA is significantly less than the persistence length (l c~2 7 nm, l p~ 50 nm), the conformation is that of a rigid rod and orientation of the dsDNA probes, relative to the gold surface, can be inferred [3,4].…”

“…In a 60 mM monovalent salt solution the Debye length is ~1.2 nm. For a more detailed description and depiction of the setup see [4].…”

Precisely Controlled Smart Polymer Scaffold for Nanoscale Manipulation of Biomolecules

“…A high gradient of the charge density at the buffer−polymer interface results in an intense electric field on the order of 100 kV/cm within a distance of approximately one Debye length from the charged surface, and this electric field imparts a large electrostatic energy to the charged nucleotides. 11 However, the thickness of the double layer, which varies from 3 nm at low ionic strength (10 mM) to about 0.6 nm at high ionic strength (300 mM), does not span the entire length of the probes, and the electrostatic interactions are confined to the base of the dsDNA probes. Hence, the electric torque that is applied to the probes is greatest for buffers of low ionic strength, where the field extends far from the charged surface.…”

“…8 The characteristic behavior of the DNA switching, such as the switching amplitude (the difference between upright and horizontal orientations) or switching dynamics are influenced by buffer pH, ionic strength, and temperature. 9,10 Additionally, the switching amplitude is influenced by conformation changes of the immobilized DNA, making it possible to detect DNA hybridization or denaturation 11 or to utilize the platform for high-throughput characterization of the DNA sequence dependence on conformation changes that are induced by DNA binding proteins such as transcription factors. 12 We present a molecular motor, which consists of short double stranded DNA (dsDNA) probes anchored on a smart polymer surface.…”