Session B8: Biophysics I

Following the Binding and Activity of Helicases with DNA Devices

Helicases are motor proteins that have the capability to separate two annealed strands of DNA or RNA. They are involved in many critical cellular processes such as DNA replication, transcription, translation, recombination and DNA repair. Much remains to be understood about the comparative roles of the 31 non-redundant DNA helicases. Here, we utilize probe-modified DNA monolayers on multiplexed gold electrodes as a sensitive recognition element of helicase binding. The electrochemical signals from these devices are highly sensitive to structural distortion of the DNA produced by the helicases. In this study we distinguished the details of three XPB helicases that belong to the Superfamily-2 of helicases. Clear changes in DNA melting temperature and duplex stability were observed upon helicase binding, shifts that could not be observed with the conventional UV-Visible absorption measurements. Binding dissociation constants were found in the range from 10-50 nM and correlated with observations of activity. ATP-stimulated DNA unwinding activity was also followed, showing exponential timescales and distinct time constants associated with conventional and molecular wrench modes of operation. These devices thus provide a sensitive measure of the structural thermodynamics and kinetics of helicase-DNA interactions.   

DNA-Inspired Self-Assembly of Nanoscale Electronic Devices

Despite remarkable examples of difficult-to-produce isolated molecular devices, the scalable nanomanufacturing of such electronics remains at a standstill due to fundamental roadblocks associated with the synthesis of large quantities of modular nanoscale circuit elements. We have introduced a methodology for mass production of nanoscale electronic elements. We have synthesized organic semiconductor moieties within DNA-like scaffolds, leveraging the rapid, efficient, and precise coupling afforded by traditional DNA bioconjugate chemistry. These DNA-inspired nanowires enable the self-assembly of active, nanoscale circuit elements at patterned electrodes. The assembly and electrical performance of these arrayed devices have been characterized through scanning microscopy techniques and custom, automated electrical probe measurements. Our unique and economically viable approach offers a new paradigm for the fabrication of nanoscale electronic circuits.   

Hyperpolarized $^{\mathrm{89}}$Y-EDTP and $^{\mathrm{89}}$Y-EDTP as potential pH-sensitive MRI agents

Transferring high thermal equilibrium polarization from electrons to nuclei, dynamic nuclear polarization (DNP) is capable of making NMR insensitive nuclei detectable at low concentration with no signal averaging. This high signal strength can be exploited in the liquid state using dissolution DNP, a process by which samples are polarized in the solid state and rapidly dissolved using a superheated solvent, creating a highly polarized, physiologically compatible liquid sample. In this study, we have investigated two ligands -- EDTP and DTPP -- as possible chelates in pH monitoring using yttrium-89. By using dissolution DNP, we have amplified the $^{\mathrm{89}}$Y NMR signals of $^{\mathrm{89}}$Y-DTPP and $^{\mathrm{89}}$Y-EDTP by \textgreater 10,000-fold and have found that both have chemical shift dependence on pH. $^{\mathrm{89}}$Y-EDTP has a chemical shift linearly dependent on pH between 5.7 and 9.15 with relatively large dispersion of almost 20 ppm, whereas $^{\mathrm{89}}$Y-DTPP exhibited a pH dependence on less than half this range. In vitro and potential in vivo studies of hyperpolarized $^{\mathrm{89}}$Y-EDTP and $^{\mathrm{89}}$Y-DTPP for pH imaging will be discussed.   

Temperature-dependent proton T1 relaxation time of water:glycerol solutions at earth's magnetic field

Although weak, the earth's magnetic field is highly uniform which is required for high-resolution nuclear magnetic resonance (NMR) spectroscopy. In this study, we have investigated the spin-lattice relaxation time (T1) of water-glycerol mixtures at earth's magnetic field. The water proton T1s at different ratios of water-glycerol contents were measured at different temperatures ranging from -20 deg C to 100 deg C. Our results indicate that pure water proton T1 increases linearly with temperature, from \textasciitilde 2 s at 20 deg C to \textasciitilde 8 s at 100 deg C. However, addition of high glycerol content in this mixture decreases the slope of this linear relationship and in fact disrupts the linearity of this behavior at low temperature. A slight turnover of the T1 slope occurs at lower temperature close to 20 deg which is indicative of the effect of changing correlation time. These results are discussed in light of the Bloembergen-Pound-Purcell (BPP) theory.   

Tracking leucine metabolism in prostate cancer cells via $^{\mathrm{13}}$C NMR spectroscopy

Nuclear magnetic resonance (NMR) spectroscopy is relatively insensitive due to the weak magnetic moments of nuclei, especially those with low gyromagnetic ratio ($\gamma )$ such as $^{\mathrm{13}}$C ($\gamma =$ 10.705 MHz/T). Fortunately, a technique known as dynamic nuclear polarization (DNP) enhances the NMR signals by transferring the much higher electron ($\gamma =$ 28,000 MHz/T) polarization to nuclei. Furthermore, the invention of dissolution DNP in 2003 has expanded DNP's large signal enhancement (more than 10,000-fold) to the biomedical realm. Significantly, dissolution DNP allows real-time tracking of metabolism via labeling the relevant substrate with $^{\mathrm{13}}$C. This study examined the real-time prostate cancer cell enzyme kinetics involved in the metabolism of [1-$^{\mathrm{13}}$C] alpha-ketoisocaproate [$\alpha $-KIC] into [1-$^{\mathrm{13}}$C] leucine and vice versa. Results of \textit{in vitro} conventional 13C NMR of cell extracts and hyperpolarized 13C NMR of living prostate cancers cells will be discussed in the context of biochemical kinetics and possible diagnostic application.