1. Interactions and dynamics of DNA, RNA and Protein channels

The translocation of DNA and RNA through membrane channels is of fundamental interest. It is an essential mechanism for numerous cell functions requiring the transport of genetic material from the nucleus and cytoplasm, and it constitutes a crucial step in the process of viral infection. We use the nanometer-size pore alpha-hemolysin, embedded in a phospholipid membrane, as a model system to study the interactions and the dynamics of polynucleotides with the protein channel. We apply an electric field across the membrane in order to draw the charged polynucleotides, one by one, through the channel, and monitor their passage dynamics during the translocation process by recording the ionic current flowing through the pore (see GIF animation #1). The ionic current provides information that allows us to discriminate between polydeoxyadenines and polydeoxycytosines in real-time (read more in PNAS paper), and measure the translocation dynamics of different polynucleotides' length, down to 4 nucleotides long (read more in PRL paper).

Semi-flexible DNA molecules are drawn through the naopore by the electric field applied across the membrane (beige). Note that the molecule has to be stretched in order to fit in the channel

Recently we stalled the translocation process and manipulated short DNA molecules inside the alpha-hemolysin channel. With this set-up, we can investigate the DNA dynamics, decoupling the effects of the driving field. We start by drawing single DNAs into the pore as explained above. As the molecules enter the channel we instantaneously turn off the electric field and measure the time required for the molecule to escape from the pore (see GIF animation #2). In this experiment the molecules are "primed" at a high energy configuration since the initial partitioning of the molecule is not favored in term of entropy. We then monitor the molecule's dynamics when it spontaneously relaxes to a lower energy configuration.

The translocation of the DNA molecules can be stalled by switching off the electrical field during their passage. The unbiased dynamics of the molecules can then be followed.

2. Stability of DNA bubbles in the bulk and on a surface.

Initiation of RNA transcription and DNA replication is associated with local melting, or "bubble formation" in the DNA duplex. The initiation occurs at DNA promoter sites that are associated with some conserved sequences (such as TATA box in eukaryotes). These sequences have the increased tendency to form local DNA bubbles, even at temperatures well below their melting point transition. The fluctuations of DNA bubbles formed in synthetic DNA have been recently studied using fluorescence correlation spectroscopy (FCS) in bulk [Bonnet et. al.]. The FCS data indicates surprisingly long timescales, on the order of 0.1 milliseconds. We extend these measurements to short DNA molecules anchored on a surface. We label our molecule with a FRET pair and record the energy transfer fluctuations one molecule at a time. Immobilization of the molecules permits us to extend the bulk dynamics from the millisecond to the second timescale, and compare the dynamics of the free molecules with the anchored ones.

Fluorescently labeled DNA molecules are anchored on a substrate and imaged using a confocal microscope. Each molecule is labeled with a FRET pair, at the bubble location. When the bubble is "closed" the donor (green plate) is partly quenched and the acceptor emits strongly (red plate). If the bubble is "open" there is no energy transfer and only the donor's emission is observed. Each spot on the red and green plates corresponds to a single DNA molecule. The scan size is 20x20 microns.

3. DNA-enzyme interactions

Under construction

4. Development of novel, stable fluorescent reporter compounds

The ability to measure the biological activity of individual biomolecules by single-molecule fluorescence methods is greatly limited by the nonideal properties of the fluorophores [Weiss, S]. Commonly used fluorophores exhibit, to varying degrees, undesirable photophysical characteristics such as spectral diffusion, changes in quantum efficiency, and the formation of long lived triplet states and long-lived "dark" states (commonly known as "blinking"). The source of these phenomena may be related to uncontrolled changes in the immediate environment of the fluorophore, or structural changes in the molecule such as "cis" to "trans" transitions. Additionally, most single-molecule measurements that rely on fluorescence probing, are further limited to short continuous observation times (up to a few seconds) due to photobleaching of the dyes. The development of better fluorescent probes that minimize these limitations is thus of crucial importance for the field of single-molecule.

In collaboration with Dr. James Foley at the Rowland Institute we have initiated a program for the rational design, synthesis and evaluation of more stable and long-lived compounds. Our focus will be on the development of organic compounds that can be easily conjugated with nucleic acids or protein and will be useful in single-molecule experiments with biomolecules.

1. Bates, M., M., Burns and A. Meller, 2003.
Dynamics of DNA molecules in a membrane channel probed by active control techniques,
Biophys. J., 84, 2366-2372 (download paper)

2. Meller, A., L. Nivon, E. Brandin, J. Golovchenko and D. Branton, 2000.
Rapid Nanopore Discrimination Between Single Polynucleotide Molecules,
Proc. Natl. Acad. Sci. 97: 1079-1084 (download paper)

3. Meller, A. L. Nivon, D. Branton, 2001.
Voltage-Driven DNA Translocations Through a Nanopore, Phys. Rev. Lett., 86:3435-39
(download paper)

4. Bonnet, G., Krichevsky, O., Libchaber, A. 2000
Unusual DNA breathing modes unravel transcription initiation specificity, Biophys. J. 78: (1) 776Plat

5. Weiss, S. 1999
Fluorescence Spectroscopy of Single Biomolecules, Science 283:1676-1683