Characterizing Conformational Fluctuations of DNA Anabel Chang, Jack Maurer, Amr Tamimi, Dylan Heussman, Pat Herbert, Peter Von Hippel and Andrew H. Marcus Printing: Department of Chemistry and Biochemistry, University of Oregon This poster is 48” wide by 36” high. RESULTS It’s designed to be printed on a INTRODUCTION FUTURE DIRECTION large Local fluctuations of the sugar-phosphate backbones of DNA (a form of DNA ‘breathing’) Single Molecule Polarization-Sweep Spectroscopy Single Photon Phase-Tagging For Single Molecule Fourier Transform play key roles in protein-DNA assembly and enzymatic function. These fluctuations Experimental Time Correlation Functions (TCFs) Electronic Spectroscopy (FTES) contribute towards the formation of DNA structures which can be recognized by proteins during replication, allowing access to the interior bases and sequence independent binding. • From the phase- and time-tagged The smFTES experimental setup. Phase Factor TCF data, the time-dependent overlap We aim to characterize the dominant structures and their fluctuations involved in spectrum of a single molecule can be Customizing the Content: functionally relevant DNA topologies, most notably ss-ds DNA fork junctions. determined.• The smFTES method can be used to monitor structural changes of a The placeholders in this Can we characterize local backbone fluctuations of ss-ds DNA forks and primer-template dimer labeled DNA construct in real junctions using photon correlation single-molecule fluorescence spectroscopy? time at the single molecule level. By Counts TCF also rotating the polarization of the Fig. 5 formatted for you. incident broadband pulses, we can Fig. 4A obtain the single molecule dichroism spectrum. placeholders to add text, or click MATERIALS & METHODS an icon to add a table, chart, Labeled DNA Replication Fork SmartArt graphic, picture or Phase Factor TCF FT DNA is “internally” labeled by insertion of a fluorescent dye into the sugar-phosphate backbone. multimedia file. Fig. 1 DNA fork constructs are prepared, which contain a (Cy3)2 dimer or a Cy3 monomer at specific positions relative to the fork junction. Counts TCF Fig. 6a Fig. 6b T Fig. 4B from text, just click the Bullets The smFTES approach can be used to track the spectral evolution of our Cy3 dimer probe. This would reveal the local conformation changes of the DNA backbones within nucleic acid-protein complexes. For example, the method could be button on the Home tab. used to study structural changes within the transcription-elongation complex (TEC) of Fig. 7, which is stable and long lived relative to experimentally accessible timescales available using this approach. A list of processes occurring in the TEC is shown in Fig. 8. along with the timescales associated with each rate of reaction. If you need more placeholders for Phase Factor TCF L. Kringle, et al., J. Chem. Phys., 2018, 148, 085101. dimer monomer labeled DNA fork Fig. 2 labeled DNA fork Transcription elongation complex Processes occurring in the TEC and associated timescales titles, construct construct Counts TCF Fig. 7 Fig. 8 make a copy of what you need and Fig. 4C Single Molecule Polarization-Sweep Spectroscopy drag it into place. PowerPoint’s Smart Guides will help you align it Fig. 3. (A) Experimental setup for Simple model of the Polarization Sweep Signals of the Cy3 Dimer and Monomer labeled DNA fork constructs: single-molecule measurements of Greive, S. J., & Von Hippel, P. H. (2005). backbone fluctuations at the DNA 𝐼 = (%&') 1 + '*% cos 𝜑 + 𝜑 𝐼 = 𝐶 [1 + 𝑣 cos 𝜑 + 𝜑 ] 𝑣 = a ‘visibility’ Thinking quantitatively about transcriptional regulation. with everything else. !"# + ,-.- + fork junction using Cy3 dimer ) %&' Nature Reviews Molecular Cell Biology, 6(3), 221–232. probes. https://doi.org/10.1038/nrm1588The constants A and B describe the overlap intensities of the plane-polarized laser with the absorption spectra of the (B) The plane polarization symmetric and anti-symmetric excitons of the dimer. The constant C is the overlap of the laser with the monomer spectrum. The Want to use your own pictures direction of a continuous wave angle 𝜑+ describes the orientation of the probe chromophore relative to the laser polarization. The time correlation functions 532 nm laser is rotated from 0 to (TCFs) in Fig. 4A – 4C compare the ‘counts’ and ‘phase-factor’ signals measured for the +1, -1, and -2 labeled DNA constructs.. 2π at 1MHz frequency. When a 3 7! 7! 3 7! *"> instead of ours? No problem! Just fluorescent photon is detected, 𝐼/-0.12= ∑4 563𝛿 𝑡 − 𝑡5 = 𝐼4 89:2;*<:/1-= = ∑563𝛿 𝑡 − 𝑡5 𝑒 " 𝑇𝐶𝐹 = 𝛿𝐼 4 ?(@*A) 𝑡 𝛿𝐼?(@*A) 𝑡 + 𝜏 ! ! ! information about the phase of CONCLUSIONS the laser polarization angle, as well as the photon arrival time, is right recorded. • Use of Cy3 monomers and dimers as probes are useful for studying structure and conformational fluctuations in ACKNOWLEDGEMENTS (C) Differential absorption in the biological systems such as dimer labeled DNA forks and junctions in the transcription-elongation complex. Change Picture. Maintain the two polarization directions is due • Presidential Undergraduate Research Scholars • UO Presidential Scholarship • Refinement of current single molecule polarization sweep will characterize the dynamics of the sugar-phosphate to the symmetric and Program antisymmetric excitons being • Professor Geraldine Richmond • UO Summit Scholarship backbones near DNA fork junctions. proportion of pictures as you resize orthogonally polarized to one • Professor Michael Haley another. • UO CAS Henry V. Howe Scholarship • Single-photon phase-tagging techniques can be used to capture structural changes occurring at the single molecule level. This information could help to elucidate bimolecular mechanisms in protein-nucleic acid complexes. by dragging a corner. Fig. 3 Phelps et al., Proc. Nat. Acad. Sci., 2013, 110, 17320-17325.