A significant part of our research involves developing cutting-edge spectroscopic and optical methods to study protein-nucleic acid interactions. The work is highly interdisciplinary as it involves elements of optical physics, molecular spectroscopy, molecular biology and biophysics.
Physics graduate student Claire Albrecht is developing a two-dimensional fluorescence spectroscopy (2DFS) instrument to study the local conformations and conformational fluctuations of fluorescent base analog substituted DNA constructs.
The methods we develop include linear and nonlinear spectroscopic techniques that are sensitive to the electronic couplings between an optical probe and its immediate biomolecular environment. The probes site-specifically label a nucleic acid construct so that spectroscopic measurements report on the local conformations of the sugar-phosphate backbones, or (in separate experiments) the nucleic acid bases. We are particularly interested in fluorescence-detection-based methods that are sensitive to the exciton-coupling of optical dimer probes, such as cyanine dimers inserted into the sugar-phosphate backbones or fluorescent base analog dimers that are site-specifically substituted for canonical bases within a nucleic acid construct. These approaches allow us to observe functionally relevant nucleic acid backbone and base-stacking conformations.
An important research goal is to develop techniques for enabling high-sensitivity measurements under low signal flux conditions. In many interesting situations, it is necessary to acquire data as rapidly as possible, while causing minimal optical damage to the sample. This requires extracting the maximum information possible from a signal that must be measured at the level of individual photons. Examples of low flux experiments include studies of fluorescence from a single molecule or a nano-structured object, or the weak illumination and subsequent detection of samples using quantum-entangled photon pairs.
Undergraduates Morgan Marsh and John Gillies taking a break from their single-molecule fluorescence experiments.
One of the approaches we have developed is microsecond-resolved single-molecule fluorescence imaging, which can provide information about the dynamics of local biomolecular conformation changes over a broad range of time scales (spanning tens-of-microseconds to several seconds). These experiments incorporate techniques to control the polarization state of the illuminating laser beam using an optical interferometer, which is synchronized to high precision digital electronics to ‘phase-tag’ individual detection events with the measured interferometer phase. These approaches allow us to achieve enhanced signal-to-noise ratios and to thereby decrease the time required to perform a measurement of a desired accuracy.
Molecular and instrumental setups for monitoring microsecond-resolved FRET and linear dichroism (LD) signals from single protein-DNA complexes. (A) The T4 gp41 helicase binds to a (dT)n loading sequence on the lagging strand of a model DNA replication fork construct. An assembled (gp41)6·gp61·DNA primosome complex can unwind the duplex region of the DNA in the presence of GTP. The strands within the dsDNA region are internally labeled with the FRET donor-acceptor iCy3 and iCy5 chromophores, respectively. (B) The sample mounted to a microscope slide is optically excited using a total internal reflection fluorescence (TIRF) geometry. The polarization state of the excitation beam is continuously varied at a frequency of 1 MHz. The p-polarization component of the incident laser points in the direction of the y-axis, and the s-polarization component is contained within the x-z plane. A pinhole aperture masks the fluorescence to transmit the image from a single molecule. Avalanche photodiodes (APDs) are used to detect photon counts from the iCy3 and iCy5 fluorescence channels. (C) Orthogonal polarization directions of the exciting laser used to measure the LD signal as seen from the perspective of the incident laser beam. Figure adapted from Phelps et al., Proc. Nat. Acad. Sci. 110, 17320-17325 (2013).
Another approach that we have developed is fluorescence-detected Fourier transform electronic coherent spectroscopy for studying the electronic couplings of optical probes in biomolecular environments. We apply these techniques to sensitively measure the linear and nonlinear absorbance spectra of exciton-coupled dimer probes in biomolecular constructs, in addition to studying the coherent excited electronic state dynamics of these systems.
Experimental apparatus used for phase-tagged photon counting (PTPC) Fourier transform spectroscopy. AOM= acousto-optic modulator, BS = beamsplitter, PMT= photomultiplier tube, APD= avalanche photodiode, NDF= neutral density filter, LPF = low-pass filter, TTL = transistor-transistor-logic voltage pulses, FPGA= field-programmable gate array. Either the lock-in (red detail) or the FPGA (blue detail) data analysis electronics could be used (depending on the signal flux), as selected by the two switches. Figure adapted from Tamimi et al., Optics Express, 28, 25194-25214 (2020).
Chemistry and Biochemistry graduate student Jack Maurer is working on a two-dimensional fluorescence spectroscopy (2DFS) instrument to study cyanine probe-labeled DNA constructs.