Translational Research
Nanodevices for Molecular Detection and Computation
New, unexpected features of the structure and mechanism of the F₁Fₒ ATP synthase, and the related A-type and V-type molecular motors continue to be revealed. This has been possible due to the inventive nature of the research labs, including our lab, who continue to devise new technologies that enable the novel features of these motors to be studied.
Nanorod Single-Molecule Assay
Gold nanorod (AuNR) assay for single-molecule rotation measurements.
We invented the use of gold nanorods for use in measuring nanoscale rotation, for which we received a few patents. The advantages over other nanoscale assays are that, unlike fluorophores, AuNR scatter high intensities of photons, and do not photobleach. This enables us to collect large amounts of data with short time resolution that can be collected for extended time periods. Due to their small size, AuNR do not exert sufficient drag on the F-type motors to become rate-limiting to rotation. As a result, rotation data reveal rate-limitations at specific mechanistic states of the motors. In addition, with these rotation probes, the majority of molecules on the slide are observed to rotate, as compared to ~5% of molecules that rotate with other assays. Consequently, the rotating molecules represent the average molecules, not the exceptions. Combined, these features provide the ability to observe much more detail of the rotary mechanism in a manner that has much improved statistical significance than other single-molecule rotation assays.
Single-Molecule Biomarker Detection Nanodevices
This is the set up for a DNA detection nanodevice.
We realized that our AuNR assay not only detected one molecule of F₁, but also detected one molecule of streptavidin, which was required to link the motor to the visible probe. We realized that we had an assay to detect a single molecule of anything if we made the assembly of the nanorod with the motor conditional to the binding of the target molecule. We have now adapted the single-molecule assay of F₁-ATPase rotation using gold nanorods for use in molecular detection of DNA sequences, proteins, and metabolites that are biomarkers for cancer and infectious diseases.
Our first proof-of-concept was to detect the presence of strands of DNA with the sequence that enables recognition of the presence of single genomes of anthrax virus for which we reached a detection limit of ~150 molecules. This increased sensitivity results when we count the number of single-molecules of F₁ rotating in the presence of ATP because each one must have bound a molecule of that DNA sequence. It is noteworthy that we virtually eliminate non-specific binding of AuNR to the slide surface using custom coatings. Non-specific binding of a detectible probe determines the limit of most detection assays.
LXR (ligation-exonuclease reaction) Technology
Ligation-Exonuclease Reaction Technology can detect single DNA molecules of a specific sequence.
We invented this in order to use the AuNR assay to detect sequence-specific target DNA. Two DNA probes, which are modified to attach to the AuNR and F₁, hybridize to adjacent positions of the target sequence. Ligase will covalently link the probes only if the sequence is perfectly complimentary to the target. Exonuclease then eliminates all mismatches, so that only the ligated DNA probes that can link to the motor and the AuNR survive.
DNA Computing
Example of a Four-City NP Complete Problem where all cities (A, B, C, D) are connected by paths (P) with varying efficiencies.
We adapted LXR for use in DNA computing where we solved a 15-city NP Complete Traveling Salesman Problem, which is the largest, most complex mathematical problem solved to date by molecular computing. These problems are difficult to solve by in silico computers because the goal is to find the most efficient path from the immense number of possible answers.
We are able to use DNA hybridization and ligation, of two city sequences, via a path sequence to make a molecular computation of the order in which cities are traveled.
Synthetic DNA strands of unique sequence are assigned either a “city” or a “path” and the goal is to compute the shortest route for the salesman to return home after visiting every city. As in enzyme kinetics, paths are added in limiting amounts proportional to the efficiency of completing that leg, while cities are present in saturating amounts. Computation results from hybridization of cities to pathways that are then ligated to form answer strands composed of a sequence of cities. The order of linked city DNA strands present in highest abundance is the optimal answer.
DNA sequences of correct answers where all cities have been visited, starting and ending with the home city. The number of sequences formed in highest abundance is the optimal answer.
Ω-qPCR nanodevices
The Ω-probe DNA strands hybridize to the sequence-specific target to form a loop that can be ligated if the sequence is an exact match. The probe contains sequence information to enable qPCR, which can only happen once the probe has been circularized by ligation.
These nanodevices use qPCR to rapidly and accurately determine the number of short DNA sequences. We invented these to increase the speed and accuracy of determining the optimal answers to Traveling Salesman Problems where the abundance of the order of any combination of city sequences can be determined simultaneously in about 25 minutes. We recently adapted these nanodevices for use in biomedical diagnostic applications.
Publications and Patents
Xiong, F. and Frasch, W. D. (2020) Probes and Methods for Measuring Tandem Repeats, US Patent 10,718,017
Xiong, F., Kuby, M., and Frasch, W. D. (2020) Accelerating DNA Computing via PLP-qPCR Answer Read out to Solve Traveling Salesman Problems in, Traveling Salesman Problems, Intech Open Press, London, peer-reviewed chapter, pp 1 - 17.
Frasch, W. D., Spetzler, D., and York, J. (2013) High Speed, High Fidelity, High Sensitivity Nucleic Acid Detection, U.S. Patent 8,530,199
Frasch, W. D., Spetzler, D., and York, J., Xiong, F. (2013) Methods for Generating a Distribution of Optimal Solutions to Nondeterministic Polynomial Optimization Problems, Patent 8,126,649
Frasch, W. D. and Chapsky, L. (2012) Polarization-Enhanced Detector with Gold Nanorods for Detecting Nanoscale Rotation and Method Therefore, Patent 8,207,323
Frasch, W. D. and Chapsky, L. (2012) Polarization-Enhanced Detector with Gold Nanorods for Detecting Nanoscale Rotational Motion and method therefor, Patent 8,192,936
Frasch, W. D., Spetzler, D., and York, J., Xiong, F. (2012) Methods for Generating a Distribution of Optimal Solutions to Nondeterministic Polynomial Optimization Problems, Patent 8,126,649
Frasch, W. D., Spetzler, D., and York, J. (2011) High Speed, High Fidelity, High Sensitivity Nucleic Acid Detection”, Patent 8,084,206
Frasch, W. D. and He, Liyan (2011) Single Molecule Detection using Molecular Motors” Patent 8,076,079
Frasch, W. D. and Chapsky, L. (2011) Polarization-Enhanced Detector with Gold Nanorods for Detecting Nanoscale Rotational Motion and method therefor, Patent 8,003,316
Xiong, F. and Frasch, W. D. (2010) “Padlock Probe-Mediated qRT-PCR for DNA Computing Answer Determination”, Natural Computing 10, 947-959.
Xiong, F., Spetzler, D., and Frasch, W. D. (2009) “Solving the Fully-Connected 15-City TSP using Probabilistic DNA Computing”, Integr. Biol., 1, 275-280.
Spetzler, D., Xiong, F., and Frasch, W. D. (2008) “Heuristic solution to a 10-City Traveling Salesman Problem Using Probabilistic DNA Computing”, LNCS 4848, 152-160.
York, J., Spetzler, D., Xiong, F., and Frasch, W. D. (2008) “Single Molecule Detection of DNA via Sequence-Specific Links between F1-ATPase Motors and Gold Nanorod Sensors”, Lab. Chip 8, 415-419. Among top-10 LOC articles accessed on-line that year. Highlighted in Chemical Biology, a Royal Society of Chemistry news magazine that provides a snapshot of the latest, most exciting, chemical biology developments.
Spetzler, D., York, J., Dobbin, C., Martin, J., Xiong, F., Ishmukhametov, R., Day, L., Yu, J., Kang, H., Porter, K., Hornung, T., and Frasch, W.D. (2007) “Recent Developments of Biomolecular Motors as On-Chip Devices using Single Molecule Techniques”, Lab. Chip 7, 1633-1643. Among top 10 most accessed LOC articles in 2007.
Spetzler, D., Xiong, F., and Frasch, W.D. (2007) “Probabilistic DNA Computing Solution to a Fully Connected 10-City Asymmetric Traveling Salesman Problem” Proc. DNA 13, 9-18.
Xiong, F., Spetzler, and Frasch, W. D. (2007) “Elimination of Secondary Structures for DNA Computing”, Proc. DNA 13, 241-249.
Chapsky, L., Frasch, W. D., Chou, C., Zenhausern, F., and Goronkin, H. (2006) Single-Molecule Detection of Biological Warfare Agents Using the F1-ATPase Biomolecular Motor, Patent 6,989,235