Individual Molecules of Dye‐Labeled DNA Act as a Reversible Two‐Color Switch upon Application of an Electric Field

Abstract

Over the past few years, molecular switches have been heavily researched in the quest for molecular electronic devices. Some switching systems operate by a conformational change in the molecule which is induced by either an electric field, [1,2] an STM (scanning tunneling microscope) tip, [3] an electrochemical reaction, [4] or light. [5] Alternatively, molecular switches can be operated nonconformationally by redox reactions [6] or a chemical binding event. [7] Biomolecules have recently been adapted for new purposes; for example, DNA has been used as part of a conducting wire, [8] a molecular machine, [9][10][11][12] a crystal template, [13] a scaffold for nanoscale construction, [14] and even as a component of a "computing machine". [15,16] Herein we show that the fluorescence of individual dye-labeled DNA molecules can be reversibly switched from green to red and vice versa upon application of an electric field.The use of nucleic acids as molecular switches is a relatively new concept and, so far, has had limited success. The use of an electric field to modulate the fluorescence of labeled DNA adsorbed onto an electrode [17] and the use of a magnetic field to control the hybridization state of DNA [18] are known. Conformational switching of DNA by the variation of buffer conditions, [11,19] the binding of adenosine to an aptamer sequence on DNA, [20] or the binding of singlestranded DNA (ssDNA) to a DNA quadruplex [9] have also recently been reported. The main limitation with the current mechanisms for DNA conformational switches is that they require bimolecular hybridization or a change of buffer, thus switching is slow. Furthermore, in some cases switching is not reversible [20] or the process creates undesirable "waste" DNA. [9,10] Our DNA switch overcomes these problems by using an electric field to alter the DNA-dye interaction which renders the switching process rapid (< 100 ms) and reversible with no by-products.Here a controllable electric field and single-molecule fluorescence detection were used to probe the switching of the fluorescent states of individual DNA molecules that were labeled with two different fluorophores at the same end of the DNA duplex. We previously utilized the very high electricfield gradients generated at the tip of a nanopipette to controllably concentrate and deliver DNA. [21][22][23] The pipettes have an inner diameter of 100 nm and the voltage drop occurs in the last few microns of the tip owing to its tapered shape. By application of a potential of 1 V between the electrode in the bath and the electrode in the pipette, a very high electric field of % 8000 V cm À1 is generated at the tip of the pipette.[22]The fluorescence of both fluorophores was detected simultaneously with either two-color direct excitation of both fluorophores or single-color excitation of the donor fluorophore only. In the latter experiments, the excited-state energy is transferred nonradiatively from a donor to an acceptor fluorophore by FRET (fluorescence resonance energy transfer). The efficiency of...

Characterization of a Single Molecule DNA Switch in Free Solution

“…The same end and opposite end duplex DNA samples used for the fluorescence lifetime experiments were prepared as previously described. 7 Briefly HPLC-purified 40-base oligonucleotide 5′-TAG TGT AAC TTA AGC CTA GGA TAA GAG CCA GTA ATC GGT A-3′ (MWG-Biotech, Ebersberg, Germany) was 3′ labeled with the fluorophore Rhodamine Green (RG); a 5′ RG labeled and unlabeled version were also purchased. Its 40-base complementary oligonucleotide with a 5′ C6 amino modifier (IBA, Go ¨ttingen, Germany) was desalted (NAP 5 column, Amersham, U.K.) and labeled with an Alexa Fluor 647 Oligonucleotide Amine Labeling Kit (Molecular Probes, Eugene, OR).…”

“…Similar fluorescence switching behavior in the DNA attached donor/acceptor system was also discovered by us, using the electric field generated inside the tip of a glass nanopipet rather than the optical method. 7 In that experiment the DNA construct has Rhodamine Green as the donor and Alexa-647 as the acceptor. Here the switching between donor-only fluorescence and acceptor-only fluorescence depends on the polarity of the electric field applied and occurred in less than 100 ms. 7 The electric field driven optical switch, which is distinct from its optical driven counterpart, does not require oxygen removal and the addition of triplet quencher.…”

“…7 In that experiment the DNA construct has Rhodamine Green as the donor and Alexa-647 as the acceptor. Here the switching between donor-only fluorescence and acceptor-only fluorescence depends on the polarity of the electric field applied and occurred in less than 100 ms. 7 The electric field driven optical switch, which is distinct from its optical driven counterpart, does not require oxygen removal and the addition of triplet quencher. Despite all these efforts, the mechanism which governs the fluorescence switching of single carbocyanine dyes is still elusive.…”

Applications of nanopipettes in bionanotechnology

“…Most DNA conformational switches presented to date are slow to switch over because either bimolecular hybridization or a change of buffer is required. The feasibility of developing a DNA switch that overcomes these problems was investigated by taking advantage of the strong local electric field near the tip of the pipette to alter the DNA-dye interaction which renders the switching process rapid (<100 ms) and reversible without by-products [15]. The DNA switch is based on a 40-bp dsDNA labelled with a donor fluorophore, Rhodamine Green, and an acceptor fluorophore, Alexa Fluor ® 647 [a Cy5 (indodicarbocyanine) derivative].…”

The scanned nanopipette: a new tool for high resolution bioimaging and controlled deposition of biomolecules

“…One particularly exciting application of the high electric field in the pipette was our finding that we could manipulate the fluorescence of a fluorophore labelled DNA on application of an electric field. 44 The DNA has a donor and acceptor fluorophore and excitation of the donor in solution leads to half the molecules emitting via their acceptor, via fluorescence resonance energy transfer, and the other half via their donor as determined by a single molecule analysis of individual DNA molecules. In the electric field in the pipette, we could switch this to 100% acceptor or donor depending on the potential applied, in less than 100 ms.…”