Dhakal Lab Website

Education

B.S., Tribhuvan University, Nepal, 2001
M.S., Tribhuvan University, Nepal, 2003
Ph.D., Kent State University, Kent, OH, 2013
Postdoctoral Research Fellow, University of Michigan, Ann Arbor, 2013-2016

Honors, awards and fellowship

Graduate Student Senate (GSS) International Travel Award, Kent State University, 2011
University Fellowship, Kent State University, 2010
Certificate of Appreciation (for excellent teaching), Kathmandu Model College, Nepal, 2005

Research interests

The research in my group spans the area of single molecule DNA nanotechnology, DNA/protein biophysics, single molecule sensing, analytical chemistry, and chemical biology. The current research focus of our group includes aptamer-based multiplex detection of disease-related small molecules and protein biomarkers, mechanical unfolding of the aptamer-target complex to quantitatively determine their mechanical stability & binding constants, and investigation of the mechanistic details of enzyme catalysis at the single molecule level. Our experimental techniques range from single molecule fluorescence microscopy to single molecule fluorescence resonance energy transfer (smFRET), optical tweezers, and molecular biology.

Single molecule sensing of disease-related biomarkers

Aptamers are short single-stranded DNA or RNA oligonucleotides capable of folding into 3D structures possessing a unique combination of loops, stems, and hairpins giving them the ability to bind small molecule ligands or protein targets with high affinity and specificity. Aptamers have become a potent rival of traditional antibodies in biotechnology because the aptamers are virtually nonimmunogenic and nontoxic in vivo, thus avoiding some unforeseen side effects. The 3D conformations of aptamers are sensitive to the microenvironment and their affinity to targets directly affects the effective dose of the drugs. Therefore, quantification of the affinity and kinetics of binding and real time data on specificity can benefit the areas of diagnostics and therapeutics. Single molecule fluorescence microscopy offers several unique advantages over conventional biochemical analysis such as it allows identification of stoichiometry by counting the number of photobleaching steps, and also enables the observation of nanometer (nm) scale dynamics of several hundred molecules simultaneously. With proper fluorescent labeling of aptamers and/or target molecules, smFRET allows real time monitoring of dynamic interactions. While the current focus of my group includes aptamer-based multiplex detection (aptasensing) of disease-related small molecules, our long-term goal is to implement the sensing strategy for the detection of cancer biomarkers in clinical samples.

Understanding how single enzymes work

Understanding the behavior of enzymes is important for understanding their role in the molecular basis of life and in a variety of diseases. Although recent single molecule experiments and theoretical analyses have proposed a diversity of conformations and cooperative conformational changes within the enzyme, there lacks a suitable technique to precisely characterize the dynamics of enzyme catalysis. By integrating optical tweezers and single molecule fluorescence microscopy on a DNA platform, my research group plan to investigate the molecular basis of enzyme catalysis, whose understanding has so far been elusive. In general, the products of enzymatic reactions are not fluorescent and therefore, the reaction has to be coupled with another reaction in which a fluorogenic substrate gets converted into a fluorescent product. The formation of the fluorescent molecules as a consequence of enzyme turnover results in intensity spikes. A precise quantification of the duration of spikes (active state) and inter-spike intervals (inactive state) allow determination of important kinetic parameters that are useful in understanding the mechanism of single-enzyme action. Integrating the powers of DNA nanotechnology and single molecule microscopy as well as optical trapping, we aim to make a lasting impact on the fields of bio-nanotechnology and chemical biology. 

Select publications

Single-molecule sensor for high-confidence detection of miRNA, Wijesinghe, K.M., Kanak, M.A., Harrell, J.C. & Dhakal, S. (2022) ACS Sensors, 7, 4, 1086-1096

Nanopore analysis as a tool for studying rapid Holliday junction dynamics and analyte binding, Ghimire, M.L., Gibbs, D.R., Mahmoud, R., Dhakal, S. & Reiner, J.E. (2022) Analytical Chemistry, 94, 28, 10027-10034

MASH FRET: A simplified approach for single-molecule multiplexing using FRET, Kaur, A., Mischa, E. & Dhakal, S. (2021) Analytical Chemistry, 93, 25, 8856-8863

Direct unfolding of RuvA-HJ complex at the single-molecule level, Gibbs, D.R., Mahmoud, R., Kaur, A. & Dhakal, S. (2021) Biophysical Journal, 120, 10, 1894-1902

A single-molecule FRET-based dynamic DNA sensor, Megalathan, A., Wijesinghe, K. M., & Dhakal, S. (2021) ACS Sensors, 6, 3, 1367-137

Single-molecule analysis of nanocircle-embedded i-motif under crowding, Megalathan, A., Wijesinghe, K. M., #Ranson, L. & Dhakal, S. (2021) J. Phys. Chem. B, 125, 9, 2193-2201

FRET-based aptasensor for the selective and sensitive detection of lysozyme, Sapkota, K. & Dhakal, S. (2020) Sensors, 20, 914

Recent applications of FRET-based multiplexed techniques, Kaur, A. & Dhakal S. (2020) Trends Anal. Chem., 123, 115777

Homologous Recombination under the Single Molecule Fluorescence Microscope, Gibbs, D.R. & Dhakal, S. (2019) Int. J. Mol. Sci., 20, 6102

Single-step FRET-based detection of femtomoles DNA, Sapkota, K., Kaur, A., Megalathan, A. Donkoh-Moore, C. & Dhakal, S. (2019) Sensors, 19, 34952.

Single molecule analysis of i-motif within self-assembled DNA duplexes and nanocircles, Megalathan, A., Cox, B.D., Wilkerson, P.D., Kaur, A., Sapkota K., Reiner, J.E., and Dhakal, S. (2019) Nucleic Acids Research, gkz565

Multiplexed nucleic acid sensing with single-molecule FRET. Kaur, A., Sapkota, K. and Dhakal, S. (2019), ACS Sensors, 4, 623-633

Build your own microscope: Step-by-step guide for building a prism-based TIRF microscope. Gibbs, D.R., Kaur, A., Megalathan, A., Sapkota, K. and Dhakal, S. (2018), Methods & Protocol, 1, 40.

Single-molecule imaging reveals conformational manipulation of Holliday junction DNA by the junction processing protein RuvA. Gibbs, D.R. & Dhakal, S. (2018), Biochemistry, 57, 3616-3624.

A bio-hybrid DNA rotor/stator nanoengine that moves along perdefined tracks. Valero, J., Pal, N., Dhakal, S., Walter, N.G. & Famulok, M. (2018), Nature Nanotechnology, 13, 496-503.

Fu, J., Yang, YR. Dhakal, S. Zhao, Z., Zhang, T., Liu, M., Walter N.G. & Yan, H. (2016) DNA Nanostructure-Scaffolded Assembly of Multienzyme Complexes. Nature Protocol, 11; 2243-2273.

Dhakal, S. Adendorff, M., Liu, M., Yan, H., Bathe, M. & Walter N.G. Rational design of DNA-actuated enzyme nanoreactors guided by single molecule analysis. Nanoscale, 2016, 8, 3125-3137.

Zhao, Z., Fu, J., Dhakal, S., Johnson-Buck, A., Liu, M., Zhang, T., Woodbury, N., Liu, Y., Walter, N.G. & Yan, H. Nano-caged enzymes with enhanced activity and stability. Nature Commun., 2016, 7, 10619.

Mallik, L.¹, Dhakal, S.¹, Nichols, J., Mahoney, J., Dosey, A. M., Jiang, S., Sunahara, R. K., Skiniotis, G. & Walter, N. G. Electron microscopic visualization of protein assemblies on flattened DNA origami. ACS Nano, 2015, 9, 7133-7141. ¹Co-first author

Widom. JR., Dhakal, S., Heinicke, LA., Walter, N. G. Single-molecule tools for enzymology, structural biology, systems biology and nanotechnology: an update. Archives of toxicology, 2014, 88, 1965-1985.

Dhakal, S., Cui, Y., Koirala, D., Ghimire, C., Kushwaha, S., Yu, Z., Yangyuoru, P. M. & Mao, H. Structural and mechanical properties of individual human telomeric G-quadruplexes in molecularly crowded solutions. Nucleic Acids Research, 2013, 41, 3915–3923.

Dhakal, S., Lafontaine, JL., Yu, Z., Koirala, D. & Mao, H. Intramolecular folding in human ILPR fragment with three C-rich repeats. PLoS ONE, 2012, 7(6): e39271. doi:10.1371/journal.pone.0039271.

Dhakal, S., Yu, Z., Konik, R., Cui, Y., Koirala, D. & Mao, H. G-quadruplex and i-motif are mutually exclusive in double stranded ILPR DNA. Biophysical Journal, 2012, 102, 2575–2584.

Dhakal, S., Mao, H., Rajendran, A., Endo, M., Sugiyama, H., Eds: Spindler L, Spada G. P, Haider S, Silva M. W. D, Fritzsche W. “G-quadruplex nanostructures probed at the single molecular level by force-based methods” in guanine quartets: structure and application. Royal Society of Chemistry Publishing, 2012, UK.

Koirala, D., Dhakal, S., Ashbridge, B., Sannohe, Y., Rodriguez, R., Sugiyama, H., Balasubramanian, S. & Mao. H. Single-Molecule Platform for Investigation of G-quadruplex and Ligand Interactions. Nature Chemistry, 2011, 3, 782-787.

Dhakal, S., Yu, Z., Konik, R., Koirala, D. & Mao, H. Formation of human ILPR G-quadruplex in dsDNA. International Review of Biophysical Chemistry, 2011, 2, N. 6.

Dhakal, S., Schonhoft, JD., Koirala, D., Yu, Z. Basu S. & Mao, H. Coexistence of an ILPR i-motif and a partially folded structure with comparable mechanical stability revealed at the single molecular level. J. Am. Chem. Soc., 2010, 132, 8991–8997.

Yu, Z., Schonhoft, JD., Dhakal, S., Bajracharya, R., Hegde, R., Basu S. & Mao, H. ILPR G-quadruplexes formed in seconds demonstrate high mechanical stabilities. J. Am. Chem. Soc., 2009, 131, 1876–1882.

Schonhoft, JD., Bajracharya, R., Dhakal, S., Yu, Z., Mao, H. & Basu S. Direct experimental evidence for quadruplex-quadruplex interaction within the human ILPR. Nucleic Acids Research, 2009, 37, 3310–3320.