Recently a variety of techniques have been developed that allow one to both manipulate and measure single molecules and in particular, biological molecules like the DNA. Attaching one end of a DNA molecule to a micron size bead and the other one to a cover slip and pulling on the bead with Optical Tweezers the tension in the DNA is measured. This system is used to monitor the reaction that takes place between DNA and proteins at the single DNA molecule level.
Significance
Biochemistry of DNA molecules is a well developed field. It allows a whole range of cut and paste procedures. The combination of these methods with nanofabrication opens the window to the use of DNA in nanotechnology. On the other hand, the sensitivity of DNA geometry to sub-pN forces allows the measurement of such forces with high precision. We plan to use the DNA manipulation technology to attach specific molecules to DNA and then investigate the forces between them and other probe molecules. Unlike in biochemistry where there is loss of information due to averaging, this technique directly monitors the reaction between two molecules. Moreover, such sensors can be miniaturized making them appropriate for integration in electronic devices.
Projects
1. Single molecule study of protein-DNA interaction
This work is performed in collaboration
with the group of Prof. A. Libchaber at Rockefeller University.[36]
Here, RecA protein is added to the solution containing tethered
DNA and it gradually polymerizes all along the DNA. The RecA-DNA
complex is 1.5 times longer than the naked DNA and accordingly,
the polymerization reaction that takes about one hour is monitored
at the single DNA molecule level by measuring the change in the
length of the tether. A nucleation
and growth model was shown to provide an accurate description
of this process.
2. Single molecule study of DNA chemical denaturation
This project consists of a study of the reaction kinetics between double stranded DNA (dsDNA) and formamide, HCONH2, in a single DNA molecule. Our single molecule studies are based on the coverslip-DNA-bead construct and on an Optical Tweezer system. l-DNA molecules (Promega, 48.5 Kb, contour length 16.5 mm) are attached to polystyrene microbeads (2.8 mm, Polysciences) at one end and to the glass coverslip at the other using a low pH protocol. The bead is trapped by the Optical Tweezer allowing to both manipulate and visualize the corresponding end of the DNA. Stretching the DNA to its maximal length with predetermined force (e.g. 8pN) one can deduce its contour length.
In this setup we monitor changes in the contour length of the DNA that result from chemical reactions occurring on the DNA. In particular, formamide is a denaturing agent whose NH2 group is replacing the DNA bases in the inter-strand hydrogen bonds. Since its volume is larger than that of a hydrogen bond, its side effect is to slightly enlarge the distance between adjacent bases. Therefore, a certain concentration of formamide on the DNA is equivalent to a particular change in its contour length. We monitor the kinetics of the reaction by measuring the length of the DNA at 1 minute intervals. We also study the changes in the kinetics resulting from applying a fixed force to the DNA. We find that applied force: i. speeds up the reaction and ii. modifies the final steady state.
3. Conductance properties of polymer-DNA hybrids
This project studies the possibility of adjusting the properties of conducting polymers using DNA. In particular, we induce chemical and structural changes in polydiacetilene (PDA) and study their effect on the conductance properties of this polymer. We replace the functional groups of PDA with DNA nucleotides and then, hybridize the resulting polymer with the complementary single stranded DNA (ssDNA). The resulting PDA-DNA hybrid will have both conductance properties due to the PDA component and also the local information from the DNA. Single molecules of such a hybrid represent smart nanowires, in the sense that: i. they are few nanometers wide, ii. they are electrically conducting and iii. they contain local information on nanometer scale which can be specifically addressed. The latter property is a form of molecular recognition and can be used to locally alter the properties of the wire. For example, restriction enzymes that cut the DNA at the location of a particular sequence can be used as a molecular switch.