Jan T. Liphardt
Plasmonics: Development of new ways for measuring molecular distances
A key problem in biophysics is the measurement of nm scale distances. In collaboration with the lab of Paul Alivisatos, we have been using characterizing the distance dependence of the plasmon resonance between two gold (or silver) nanoparticles. Unlike conventional dyes, noble metal nanoparticles do not blink or bleach, making it possible to track them, or use them to measure distances, for arbitrary durations. For an overview of our ongoing plasmon resonance work, please see the meeting report in Science 308 (2005).
Color effect on directed assembly of DNA-functionalized gold and silver nanoparticles. (a) The basic principle: nanoparticles functionalized with streptavidin are attached to the glass surface coated with BSA-biotin (left). Then, a second particle is attached to the first particle (center), again via biotin-streptavidin binding (right). The biotin on the second particle is covalently linked to the 3' end of a 33 base pair long ssDNA strand bound to the particle via a thiol group at the 5' end. Inset: principle of transmission darkfield microscopy. (b) Single silver particles appear blue (left) and particle pairs blue-green (right). The orange dot in the bottom comes from an aggregate of more than two particles. (c) Single gold particles appear green (left), gold particle pairs, orange (right). Inset: representative transmission electron microscopy image of a particle pair to show that each colored dot comes from light scatted from two closely lying particles, which cannot be separated optically. (d) Representative scattering spectra of single particles and particle pairs for silver (top) and gold (bottom). Silver particles show a larger spectral shift (102 nm) than gold particles (23 nm), stronger light scattering and a smaller plasmon line width. Gold, however, is chemically more stable and is more easily conjugated to biomolecules via -SH, -NH2 or -CN functional groups.
Kinetics of RNA folding; Mechanical properties of RNA
RNA molecules perform many activities in the cell, including structural scaffolding, information transfer (e.g. mRNA and tRNA), and catalysis (e.g. catalysis of the peptide bond during protein synthesis). We would like to learn how RNA folds, and characterize the mechanical properties of RNA, such as resistence to mechanical stresses and strains.
Single-ion channel and single RNA hairpin unfolding kinetics. The opening and closing of an ion channel by an electric field is a highly cooperative event, leading to all-or-none fluctuations between the closed and open states (upper panel). The equation in the inset relates the opening rate constant a to the energy required to open the channel, where ΔG is the height of the activation energy barrier at zero voltage, e is the elementary charge, and z is the number of gating charges that move in the electric field. The trace on the right illustrates a typical recording of the activity of a single ion channel. The all-or-none unfolding of an RNA hairpin (lower panel) can be triggered by a mechanical stretching force, F, applied to the 3' and 5' ends. A mechanical force increases the probability of unfolding by exponentially speeding up the rate of unfolding a and decreasing the refolding rate b, much as predicted by G. I. Bell in 1978. The trace on the right shows a recording of the all-or-none changes in length from a single RNA hairpin [The beautiful illustration is not mine, but was taken from Fernandez et al., and shows data from Liphardt et al.].
Nonequilibrium Statistical Mechanics
One of the basic problems in biophysics is the following. Frequently, one wishes to obtain the equilibrium free energy of a reaction such as RNA unfolding, but it turns out to be difficult (or even impossible) to perform the reaction quasi-statically. Instead, the system relaxes only very slowly and the reaction is irreversible, and only an upper bound on the free energy (the total work) can be obtained. In 1997, however, C. Jarzynski proved an equality that relates the irreversible work to the equilibrium free energy, <e-W/kT> = e-ΔG/kT.
We have recently tested this equality by using it to recover the equilibrium free energy of unfolding a single RNA molecule from nonequilibrium experiments. We are now extending this work by exploring different regimes (near and far-from-equilibrium) and the convergence properties of Jarzynski's equality.