36 results on '"Alexander Johnson-Buck"'
Search Results
2. Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion
- Author
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Zhao Zhao, Jinglin Fu, Soma Dhakal, Alexander Johnson-Buck, Minghui Liu, Ting Zhang, Neal W. Woodbury, Yan Liu, Nils G. Walter, and Hao Yan
- Subjects
Science - Abstract
Cells compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here, the authors describe a self-assembly approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability, and observe an inversed correlation between the protein size and the activity enhancement.
- Published
- 2016
- Full Text
- View/download PDF
3. Attomolar Sensitivity in Single Biomarker Counting upon Aqueous Two-Phase Surface Enrichment
- Author
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Zi Li, Molly McNeely, Erin Sandford, Muneesh Tewari, Alexander Johnson-Buck, and Nils G. Walter
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Fluid Flow and Transfer Processes ,Limit of Detection ,Nucleic Acids ,Process Chemistry and Technology ,Fluorescence Resonance Energy Transfer ,Humans ,Nanotechnology ,Bioengineering ,Instrumentation ,Biomarkers - Abstract
From longstanding techniques like enzyme-linked immunosorbent assay (ELISA) to modern next-generation sequencing, many of the most sensitive and specific biomarker detection assays require capture of the analyte at a surface. While surface-based assays provide advantages, including the ability to reduce background by washing away excess reagents and/or increase specificity through analyte-specific capture probes, the limited efficiency of capture from dilute solution often restricts assay sensitivity to the femtomolar-to-nanomolar range. Although assays for many nucleic acid analytes can decrease limits of detection (LODs) to the subfemtomolar range using polymerase chain reaction, such amplification may introduce biases, errors, and an increased risk of sample cross-contamination. Furthermore, many analytes cannot be amplified easily, including short nucleic acid fragments, epigenetic modifications, and proteins. To address the challenge of achieving subfemtomolar LODs in surface-based assays without amplification, we exploit an aqueous two-phase system (ATPS) to concentrate target molecules in a smaller-volume phase near the assay surface, thus increasing capture efficiency compared to passive diffusion from the original solution. We demonstrate the utility of ATPS-enhanced capture via single molecule recognition through equilibrium Poisson sampling (SiMREPS), a microscopy technique previously shown to possess99.9999% detection specificity for DNA mutations but an LOD of only ∼1-5 fM. By combining ATPS-enhanced capture with a Förster resonance energy transfer (FRET)-based probe design for rapid data acquisition over many fields of view, we improve the LOD ∼ 300-fold to10 aM for an
- Published
- 2022
4. A guide to accelerated direct digital counting of single nucleic acid molecules by FRET-based intramolecular kinetic fingerprinting
- Author
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Shankar Mandal, Nils G. Walter, Alexander Johnson-Buck, and Kunal Khanna
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Detection limit ,0303 health sciences ,Total internal reflection fluorescence microscope ,Chemistry ,030302 biochemistry & molecular biology ,Mutant ,Fluorescence ,Article ,General Biochemistry, Genetics and Molecular Biology ,Kinetics ,MicroRNAs ,03 medical and health sciences ,Förster resonance energy transfer ,Limit of Detection ,Nucleic Acids ,Intramolecular force ,Fluorescence Resonance Energy Transfer ,Nucleic acid ,Biophysics ,Nanotechnology ,Molecule ,Molecular Biology ,030304 developmental biology - Abstract
Cell-free nucleic acids (cfNAs) such as short non-coding microRNA (miRNA) and circulating tumor DNA (ctDNA) that reside in bodily fluids have emerged as potential cancer biomarkers. Methods for the rapid, highly specific, and sensitive monitoring of cfNAs in biofluids have, therefore, become increasingly attractive as clinical diagnosis tools. As a next generation technology, we provide a practical guide for an amplification-free, single molecule Forster resonance energy transfer (smFRET)-based kinetic fingerprinting approach termed intramolecular single molecule recognition through equilibrium Poisson sampling, or iSiMREPS, for the rapid detection and counting of miRNA and mutant ctDNA with virtually unlimited specificity and single molecule sensitivity. iSiMREPS utilizes a pair of fluorescent detection probes, wherein one probe immobilizes the target molecules on the surface, and the other probe transiently and reversibly binds to the target to generate characteristic time-resolved fingerprints as smFRET signal that are detected in a total internal reflection fluorescence microscope. Analysis of these kinetic fingerprints enables near-perfect discrimination between specific binding to target molecules and nonspecific background binding. By accelerating kinetic fingerprinting using the denaturant formamide and reducing background signals by removing target-less probes from the surface via toehold-mediated strand displacement, iSiMREPS has been demonstrated to count miR-141 and EGFR exon 19 deletion ctDNA molecules with a limit of detection (LOD) of ~1 and 3 fM, respectively, as well as mutant allele fractions as low as 0.0001%, during a standard acquisition time of only ~10 s per field of view. In this review, we provide a detailed roadmap for implementing iSiMREPS more broadly in research and clinical diagnostics, combining rapid analysis, high specificity, and high sensitivity.
- Published
- 2022
5. Single-molecule mechanical fingerprinting with DNA nanoswitch calipers
- Author
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Andrew Ward, Darren Yang, Wesley P. Wong, Serkan Cabi, Elisha Krieg, Bhavik Nathwani, Prakash Shrestha, Yi Luo, Toma E. Tomov, William M. Shih, James I. MacDonald, Hans T. Bergal, and Alexander Johnson-Buck
- Subjects
chemistry.chemical_classification ,Magnetic tweezers ,Materials science ,Biomolecule ,Biomedical Engineering ,Force spectroscopy ,Bioengineering ,Nanotechnology ,Condensed Matter Physics ,Proteomics ,Atomic and Molecular Physics, and Optics ,chemistry.chemical_compound ,Optical tweezers ,chemistry ,DNA nanotechnology ,Nanobiotechnology ,General Materials Science ,Electrical and Electronic Engineering ,DNA - Abstract
Decoding the identity of biomolecules from trace samples is a longstanding goal in the field of biotechnology. Advances in DNA analysis have substantially affected clinical practice and basic research, but corresponding developments for proteins face challenges due to their relative complexity and our inability to amplify them. Despite progress in methods such as mass spectrometry and mass cytometry, single-molecule protein identification remains a highly challenging objective. Towards this end, we combine DNA nanotechnology with single-molecule force spectroscopy to create a mechanically reconfigurable DNA nanoswitch caliper capable of measuring multiple coordinates on single biomolecules with atomic resolution. Using optical tweezers, we demonstrate absolute distance measurements with angstrom-level precision for both DNA and peptides, and using multiplexed magnetic tweezers, we demonstrate quantification of relative abundance in mixed samples. Measuring distances between DNA-labelled residues, we perform single-molecule fingerprinting of synthetic and natural peptides, and show discrimination, within a heterogeneous population, between different posttranslational modifications. DNA nanoswitch calipers are a powerful and accessible tool for characterizing distances within nanoscale complexes that will enable new applications in fields such as single-molecule proteomics. DNA nanoswitch calipers can measure distances within single molecules with atomic resolution. Applied to single-molecule proteomics, they can enable the identification and quantification of molecules in trace samples via mechanical fingerprinting.
- Published
- 2021
6. Direct kinetic fingerprinting and digital counting of single protein molecules
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Tanmay Chatterjee, Ning Liu, Achim Knappik, Sung Won Choi, Evan Thrush, Kenneth J. Oh, Erin Sandford, Muneesh Tewari, Alexander Johnson-Buck, Nils G. Walter, and William Strong
- Subjects
Analyte ,Quantitative proteomics ,Enzyme-Linked Immunosorbent Assay ,Antibodies ,Epitope ,Matrix (chemical analysis) ,Antigen ,Antibody Specificity ,Limit of Detection ,Humans ,Nanotechnology ,Detection limit ,Multidisciplinary ,biology ,Chemistry ,Proteins ,Reproducibility of Results ,Biological Sciences ,Single Molecule Imaging ,Kinetics ,Biochemistry ,biology.protein ,Target protein ,Antibody ,Biomarkers ,Protein Binding - Abstract
The sensitive and accurate quantification of protein biomarkers plays important roles in clinical diagnostics and biomedical research. Sandwich ELISA and its variants accomplish the capture and detection of a target protein via two antibodies that tightly bind at least two distinct epitopes of the same antigen and have been the gold standard for sensitive protein quantitation for decades. However, existing antibody-based assays cannot distinguish between signal arising from specific binding to the protein of interest and nonspecific binding to assay surfaces or matrix components, resulting in significant background signal even in the absence of the analyte. As a result, they generally do not achieve single-molecule sensitivity, and they require two high-affinity antibodies as well as stringent washing to maximize sensitivity and reproducibility. Here, we show that surface capture with a high-affinity antibody combined with kinetic fingerprinting using a dynamically binding, low-affinity fluorescent antibody fragment differentiates between specific and nonspecific binding at the single-molecule level, permitting the direct, digital counting of single protein molecules with femtomolar-to-attomolar limits of detection (LODs). We apply this approach to four exemplary antigens spiked into serum, demonstrating LODs 55- to 383-fold lower than commercially available ELISA. As a real-world application, we establish that endogenous interleukin-6 (IL-6) can be quantified in 2-µL serum samples from chimeric antigen receptor T cell (CAR-T cell) therapy patients without washing away excess serum or detection probes, as is required in ELISA-based approaches. This kinetic fingerprinting thus exhibits great potential for the ultrasensitive, rapid, and streamlined detection of many clinically relevant proteins.
- Published
- 2020
7. DNA-Templated Timer Probes for Multiplexed Sensing
- Author
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Yingnan Deng, Changyuan Yu, Qianqian Han, Liang Ma, Alexander Johnson-Buck, and Xin Su
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DNA, Bacterial ,Fluorophore ,DNA polymerase ,Computer science ,Bioengineering ,02 engineering and technology ,Polymerase Chain Reaction ,Multiplexing ,chemistry.chemical_compound ,Nucleic Acids ,General Materials Science ,Fluorescent Dyes ,Bacteria ,biology ,Mechanical Engineering ,General Chemistry ,Processivity ,021001 nanoscience & nanotechnology ,Condensed Matter Physics ,genomic DNA ,Crosstalk (biology) ,chemistry ,Nucleic acid ,biology.protein ,DNA Probes ,0210 nano-technology ,Biological system ,DNA - Abstract
Simultaneous analysis based on encoded fluorophores suffers from potential crosstalk between fluorophores and the limited number of colors that can be practically resolved. Inspired by nontrivial temporal patterns in living organisms, we developed a DNA-templated probe by utilizing DNA polymerase (DNAP) for multiplexed detection of nucleic acids. These probes use differential delay times of signaling by a DNAP-mediated extension to distinguish different targets, which serve as the primers. Taking advantage of the high processivity and the controllable kinetics of DNAP, we find that multiplexed detection can be achieved in homogeneous solution using a single fluorophore. As a proof of concept, we developed assays for genomic DNA from four different bacteria. In addition, we designed and implemented probes to undergo a single oscillation in signal as an alternative way for multiplexing. We anticipate this approach will find broad applications not only in sensing but also in synthetic DNA nanosystems.
- Published
- 2020
8. Highly Sensitive Protein Detection by Aptamer-Based Single-Molecule
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Tanmay Chatterjee, Alexander Johnson-Buck, and Nils G. Walter
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History ,Polymers and Plastics ,Business and International Management ,Industrial and Manufacturing Engineering - Published
- 2022
9. Highly sensitive protein detection by aptamer-based single-molecule kinetic fingerprinting
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Tanmay Chatterjee, Alexander Johnson-Buck, and Nils G. Walter
- Subjects
Vascular Endothelial Growth Factor A ,Interleukin-8 ,SELEX Aptamer Technique ,Biomedical Engineering ,Biophysics ,Antibodies, Monoclonal ,Reproducibility of Results ,Biosensing Techniques ,General Medicine ,Aptamers, Nucleotide ,Ligands ,Nucleic Acids ,Electrochemistry ,Humans ,Biomarkers ,Biotechnology - Abstract
Sensitive assays of protein biomarkers play critical roles in clinical diagnostics and biomedical research. Such assays typically employ immunoreagents such as monoclonal antibodies that suffer from several drawbacks, including relatively tedious production, significant batch-to-batch variability, and challenges in site-specific, stoichiometric modification with fluorophores or other labels. One proposed alternative to such immunoreagents, nucleic acid aptamers generated by systematic evolution of ligand by exponential enrichment (SELEX), can be chemically synthesized with much greater ease, precision, and reproducibility than antibodies. However, most aptamers exhibit relatively poor affinity, yielding low sensitivity in the assays employing them. Recently, single molecule recognition through equilibrium Poisson sampling (SiMREPS) has emerged as a platform for detecting proteins and other biomarkers with high sensitivity without requiring high-affinity detection probes. In this manuscript, we demonstrate the applicability and advantages of aptamers as detection probes in SiMREPS as applied to two clinically relevant biomarkers, VEGF
- Published
- 2022
10. Highly specific, amplification-free, single-molecule counting of rare methylated DNA cancer biomarkers
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Liuhan Dai, Alexander Johnson-Buck, Muneesh Tewari, and Nils G. Walter
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Biophysics - Published
- 2022
11. In situ detection of single microRNA molecules by transient hybridization utilizing FRET-based probes
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Karen Montoya, Lidan Li, Robb S. Welty, Alexander Johnson-Buck, and Nils G. Walter
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Biophysics - Published
- 2022
12. Single-molecule mechanical fingerprinting with DNA nanoswitch calipers
- Author
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Prakash, Shrestha, Darren, Yang, Toma E, Tomov, James I, MacDonald, Andrew, Ward, Hans T, Bergal, Elisha, Krieg, Serkan, Cabi, Yi, Luo, Bhavik, Nathwani, Alexander, Johnson-Buck, William M, Shih, and Wesley P, Wong
- Subjects
Spectrum Analysis ,Calibration ,Nanotechnology ,Reproducibility of Results ,Amino Acid Sequence ,DNA ,Peptides ,Protein Processing, Post-Translational ,Single Molecule Imaging - Abstract
Decoding the identity of biomolecules from trace samples is a longstanding goal in the field of biotechnology. Advances in DNA analysis have substantially affected clinical practice and basic research, but corresponding developments for proteins face challenges due to their relative complexity and our inability to amplify them. Despite progress in methods such as mass spectrometry and mass cytometry, single-molecule protein identification remains a highly challenging objective. Towards this end, we combine DNA nanotechnology with single-molecule force spectroscopy to create a mechanically reconfigurable DNA nanoswitch caliper capable of measuring multiple coordinates on single biomolecules with atomic resolution. Using optical tweezers, we demonstrate absolute distance measurements with ångström-level precision for both DNA and peptides, and using multiplexed magnetic tweezers, we demonstrate quantification of relative abundance in mixed samples. Measuring distances between DNA-labelled residues, we perform single-molecule fingerprinting of synthetic and natural peptides, and show discrimination, within a heterogeneous population, between different posttranslational modifications. DNA nanoswitch calipers are a powerful and accessible tool for characterizing distances within nanoscale complexes that will enable new applications in fields such as single-molecule proteomics.
- Published
- 2020
13. Automatic classification and segmentation of single-molecule fluorescence time traces with deep learning
- Author
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Leyou Zhang, Nils G. Walter, Alexander Johnson-Buck, and Jieming Li
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0301 basic medicine ,Databases, Factual ,Computer science ,Science ,General Physics and Astronomy ,010402 general chemistry ,01 natural sciences ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,Deep Learning ,Single-molecule biophysics ,Machine learning ,False positive paradox ,Fluorescence Resonance Energy Transfer ,Segmentation ,lcsh:Science ,Hidden Markov model ,Total internal reflection microscopy ,Multidisciplinary ,Artificial neural network ,business.industry ,Deep learning ,Pattern recognition ,General Chemistry ,Thresholding ,Single Molecule Imaging ,0104 chemical sciences ,ErbB Receptors ,030104 developmental biology ,Microscopy, Fluorescence ,Poisson sampling ,lcsh:Q ,Artificial intelligence ,Neural Networks, Computer ,business ,Transfer of learning ,Algorithms ,Software - Abstract
Traces from single-molecule fluorescence microscopy (SMFM) experiments exhibit photophysical artifacts that typically necessitate human expert screening, which is time-consuming and introduces potential for user-dependent expectation bias. Here, we use deep learning to develop a rapid, automatic SMFM trace selector, termed AutoSiM, that improves the sensitivity and specificity of an assay for a DNA point mutation based on single-molecule recognition through equilibrium Poisson sampling (SiMREPS). The improved performance of AutoSiM is based on accepting both more true positives and fewer false positives than the conventional approach of hidden Markov modeling (HMM) followed by hard thresholding. As a second application, the selector is used for automated screening of single-molecule Förster resonance energy transfer (smFRET) data to identify high-quality traces for further analysis, and achieves ~90% concordance with manual selection while requiring less processing time. Finally, we show that AutoSiM can be adapted readily to novel datasets, requiring only modest Transfer Learning., Traces from single-molecule fluorescence microscopy (SMFM) experiments exhibit photophysical artifacts that typically make analysis time-consuming. Here, the authors have developed an easily accessible software, AutoSiM, for two distinct applications of deep learning to the efficient processing of SMFM time traces.
- Published
- 2020
14. Exploring the speed limit of toehold exchange with a cartwheeling DNA acrobat
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Nils G. Walter, Alexander Johnson-Buck, Hao Yan, William M. Shih, Jieming Li, and Yuhe R. Yang
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0301 basic medicine ,Computer science ,Oligonucleotide ,Computation ,Biomedical Engineering ,Bioengineering ,DNA walker ,02 engineering and technology ,021001 nanoscience & nanotechnology ,Condensed Matter Physics ,Atomic and Molecular Physics, and Optics ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,chemistry ,DNA nanotechnology ,General Materials Science ,Electrical and Electronic Engineering ,0210 nano-technology ,Biological system ,DNA - Abstract
Dynamic DNA nanotechnology has yielded nontrivial autonomous behaviours such as stimulus-guided locomotion, computation and programmable molecular assembly. Despite these successes, DNA-based nanomachines suffer from slow kinetics, requiring several minutes or longer to carry out a handful of operations. Here, we pursue the speed limit of an important class of reactions in DNA nanotechnology—toehold exchange—through the single-molecule optimization of a novel class of DNA walker that undergoes cartwheeling movements over a field of complementary oligonucleotides. After optimizing this DNA ‘acrobat’ for rapid movement, we measure a stepping rate constant approaching 1 s−1, which is 10- to 100-fold faster than prior DNA walkers. Finally, we use single-particle tracking to demonstrate movement of the walker over hundreds of nanometres within 10 min, in quantitative agreement with predictions from stepping kinetics. These results suggest that substantial improvements in the operating rates of broad classes of DNA nanomachines utilizing strand displacement are possible. Single-molecule optimization leads to a cartwheeling DNA walker with a more than tenfold improved stepping rate.
- Published
- 2018
15. Single-Molecule Clocks Controlled by Serial Chemical Reactions
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William M. Shih and Alexander Johnson-Buck
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0301 basic medicine ,Chemistry ,Mechanical Engineering ,Ensemble averaging ,Analytical chemistry ,Bioengineering ,02 engineering and technology ,General Chemistry ,021001 nanoscience & nanotechnology ,Condensed Matter Physics ,Chemical reaction ,Thresholding ,Article ,Dissociation (chemistry) ,03 medical and health sciences ,Chemical clock ,030104 developmental biology ,DNA nanotechnology ,Molecule ,General Materials Science ,0210 nano-technology ,Iodine clock reaction ,Biological system - Abstract
Chemical clocks usually achieve well-defined temporal delays through concentration thresholding coupled to the production, degradation, activation, or inhibition of downstream effectors. In this way, the stochastic dynamics of many individual molecules yield essentially deterministic bulk behavior through ensemble averaging. As a result, their temporal evolution is governed by ensemble dynamics rather than by the behavior of an individual molecule or complex. Here, we present a general approach for the design of single-molecule clocks that permits quasi-deterministic control over the lifetime of single molecular interactions without any external synchronization. By coupling the dissociation of a bimolecular complex to a series of irreversible chemical steps, we interpose a well-defined time delay between binding and dissociation. The number and speed of irreversible steps can be varied to systematically tune both the lifetimes of complexes and the precision of the time delay, raising the prospect of localized timekeeping in nanoscale systems and devices.
- Published
- 2017
16. Rapid kinetic fingerprinting of single nucleic acid molecules by a FRET-based dynamic nanosensor
- Author
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Shankar Mandal, Nils G. Walter, Aaron T. Blanchard, Kunal Khanna, Muneesh Tewari, and Alexander Johnson-Buck
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Biomedical Engineering ,Biophysics ,Biosensing Techniques ,02 engineering and technology ,Computational biology ,01 natural sciences ,Article ,DNA sequencing ,chemistry.chemical_compound ,Nanosensor ,Nucleic Acids ,Fluorescence Resonance Energy Transfer ,Electrochemistry ,Fluorescence microscope ,Digital polymerase chain reaction ,Detection limit ,Chemistry ,010401 analytical chemistry ,General Medicine ,021001 nanoscience & nanotechnology ,Fluorescence ,Single Molecule Imaging ,0104 chemical sciences ,Kinetics ,MicroRNAs ,Förster resonance energy transfer ,Nucleic acid ,0210 nano-technology ,DNA ,Biotechnology - Abstract
Biofluid-derived cell-free nucleic acids such as microRNAs (miRNAs) and circulating tumor-derived DNAs (ctDNAs) have emerged as promising disease biomarkers. Conventional detection of these biomarkers by digital PCR and next generation sequencing, although highly sensitive, requires time-consuming extraction and amplification steps that also increase the risk of sample loss and cross-contamination. To achieve the direct, rapid, and amplification-free detection of miRNAs and ctDNAs with near-perfect specificity and single-molecule level sensitivity, we herein designed a single-molecule kinetic fingerprinting assay, termed intramolecular single-molecule recognition through equilibrium Poisson sampling (iSiMREPS). iSiMREPS exploits a dynamic DNA nanosensor comprising a surface anchor and a pair of fluorescent detection probes: one probe captures a target molecule onto the surface, while the other transiently interrogates the target to generate kinetic fingerprints by intramolecular single-molecule Forster resonance energy transfer (smFRET) that are recorded by single-molecule fluorescence microscopy and identify the target after kinetic filtering and data analysis. We optimize the sensor design, use formamide to further accelerate the fingerprinting kinetics, and maximize sensitivity by removing non-target-bound probes using toehold-mediated strand displacement to reduce background. We show that iSiMREPS can detect, in as little as 10 s, two distinct, promising cancer biomarkers—miR-141 and a common EGFR exon 19 deletion—reaching a limit of detection (LOD) of ~3 fM and a mutant allele fraction among excess wild-type as low as 1 in 1 million, or 0.0001%. We anticipate that iSiMREPS will find utility in research and clinical diagnostics based on its features of rapid detection, high specificity, sensitivity, and generalizability.
- Published
- 2021
17. Single-Molecule Fret Based Intra-Molecular Kinetic Fingerprinting for Ultrafast and High-Confidence Counting of Mutant DNA
- Author
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Shankar Mandal, Muneesh Tewari, Alexander Johnson-Buck, Kunal Khanna, and Nils G. Walter
- Subjects
chemistry.chemical_compound ,chemistry ,Mutant ,Biophysics ,Single-molecule FRET ,Kinetic energy ,Ultrashort pulse ,DNA - Published
- 2021
18. Iterative Kinetic Proofreading for High-Specificity DNA Sequence Discrimination
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Nils G. Walter, Chandler Petersen, and Alexander Johnson-Buck
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Chemistry ,Biophysics ,Computational biology ,Kinetic proofreading ,DNA sequencing - Published
- 2021
19. Highly Sensitive Protein Quantification by Direct Kinetic Fingerprinting of Single Protein Molecules
- Author
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Tanmay Chatterjee, Nils G. Walter, Kenneth J. Oh, Ning Liu, Muneesh Tewari, Achim Knappik, Sung Won Choi, Alexander Johnson-Buck, Erin Sandford, Willium B. Strong, and Evan Thrush
- Subjects
Protein molecules ,Chemistry ,Quantitative proteomics ,Biophysics ,Kinetic energy ,Highly sensitive - Published
- 2021
20. A two-layer assay for single-nucleotide variants utilizing strand displacement and selective digestion
- Author
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Xin Su, Tongbo Wu, Alexander Johnson-Buck, Yingjie Yu, and Lidan Li
- Subjects
Exonucleases ,Models, Molecular ,DNA Mutational Analysis ,Population ,Mutant ,Biomedical Engineering ,Biophysics ,Biosensing Techniques ,02 engineering and technology ,Computational biology ,Biology ,010402 general chemistry ,Polymorphism, Single Nucleotide ,01 natural sciences ,chemistry.chemical_compound ,Nucleic acid thermodynamics ,Electrochemistry ,Humans ,Point Mutation ,education ,Gene ,Fluorescent Dyes ,Genetics ,education.field_of_study ,Nucleotides ,Point mutation ,DNA ,General Medicine ,021001 nanoscience & nanotechnology ,Bacteriophage lambda ,0104 chemical sciences ,Spectrometry, Fluorescence ,chemistry ,Nucleic acid ,0210 nano-technology ,Biotechnology - Abstract
Point mutations have emerged as prominent biomarkers for disease diagnosis, particularly in the case of cancer. Discovering single-nucleotide variants (SNVs) is also of great importance for the identification of single-nucleotide polymorphisms within the population. The competing requirements of thermodynamic stability and specificity in conventional nucleic acid hybridization probes make it challenging to achieve highly precise detection of point mutants. Here, we present a fluorescence-based assay for low-abundance mutation detection based on toehold-mediated strand displacement and nuclease-mediated strand digestion that enables highly precise detection of point mutations. We demonstrate that this combined assay provides 50-1000-fold discrimination (mean value: 255) between all possible single-nucleotide mutations and their corresponding wild-type sequence for a model DNA target. Using experiments and kinetic modeling, we investigate probe properties that obtain additive benefits from both strand displacement and nucleolytic digestion, thus providing guidance for the design of enzyme-mediated nucleic acid assays in the future.
- Published
- 2016
21. CpG Methylation Detection with Single-Molecule Recognition through Equilibrium Poisson Sampling
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Nils G. Walter, Muneesh Tewari, Liuhan Dai, and Alexander Johnson-Buck
- Subjects
Chemistry ,DNA methylation ,Biophysics ,Poisson sampling ,Molecule ,Computational biology - Published
- 2020
22. Ultraspecific analyte detection by direct kinetic fingerprinting of single molecules
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Kunal Khanna, Zi Li, Nils G. Walter, Tanmay Chatterjee, Karen Montoya, Alexander Johnson-Buck, and Muneesh Tewari
- Subjects
Analyte ,biology ,Chemistry ,DNA polymerase ,010401 analytical chemistry ,Computational biology ,01 natural sciences ,Article ,0104 chemical sciences ,Analytical Chemistry ,law.invention ,law ,biology.protein ,Nucleic acid ,Disease biomarker ,Molecule ,Spectroscopy ,Polymerase chain reaction - Abstract
The detection and quantification of biomarkers have numerous applications in biological research and medicine. The most widely used methods to detect nucleic acids require amplification via the polymerase chain reaction (PCR). However, errors arising from the imperfect copying fidelity of DNA polymerases, limited specificity of primers, and heat-induced damage reduce the specificity of PCR-based methods, particularly for single-nucleotide variants. Furthermore, not all analytes can be amplified efficiently. While amplification-free methods avoid these pitfalls, the specificity of most such methods is strictly constrained by probe binding thermodynamics, which for example hampers detection of rare somatic mutations. In contrast, single-molecule recognition through equilibrium Poisson sampling (SiMREPS) provides ultraspecific detection with single-molecule and single-nucleotide sensitivity by monitoring the repetitive interactions of a fluorescent probe with surface-immobilized targets. In this review, we discuss SiMREPS in comparison with other analytical approaches, and describe its utility in quantifying a range of nucleic acids and other analytes.
- Published
- 2020
23. Ultraspecific and Amplification-Free Quantification of Mutant DNA by Single-Molecule Kinetic Fingerprinting
- Author
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Stephen L Hayward, Nils G. Walter, Paul E. Lund, Muneesh Tewari, Alexander Johnson-Buck, and Qing Kang
- Subjects
0301 basic medicine ,Mutant ,EGFR T790M ,Computational biology ,010402 general chemistry ,medicine.disease_cause ,01 natural sciences ,Biochemistry ,Polymerase Chain Reaction ,Catalysis ,DNA sequencing ,Article ,law.invention ,03 medical and health sciences ,chemistry.chemical_compound ,Colloid and Surface Chemistry ,law ,medicine ,Molecule ,Humans ,Polymerase chain reaction ,Fluorescent Dyes ,Mutation ,General Chemistry ,DNA ,Orders of magnitude (mass) ,0104 chemical sciences ,ErbB Receptors ,Kinetics ,030104 developmental biology ,chemistry ,Microscopy, Fluorescence ,Drug Resistance, Neoplasm - Abstract
Conventional techniques for detecting rare DNA sequences require many cycles of PCR amplification for high sensitivity and specificity, potentially introducing significant biases and errors. While amplification-free methods exist, they rarely achieve the ability to detect single molecules, and their ability to discriminate between single-nucleotide variants is often dictated by the specificity limits of hybridization thermodynamics. Here we show that a direct detection approach using single-molecule kinetic fingerprinting can surpass the thermodynamic discrimination limit by three orders of magnitude, with a dynamic range of up to five orders of magnitude with optional super-resolution analysis. This approach detects mutations as subtle as the drug resistance-conferring cancer mutation EGFR T790M (a single C→T substitution) with an estimated specificity of 99.99999%, surpassing even the leading PCR-based methods and enabling detection of 1 mutant molecule in a background of at least 1 million wild-type molecules. This level of specificity revealed rare, heat-induced cytosine deamination events that introduce false positives in PCR-based detection, but which can be overcome in our approach through milder thermal denaturation and enzymatic removal of damaged nucleobases.
- Published
- 2018
24. A guide to nucleic acid detection by single-molecule kinetic fingerprinting
- Author
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Muneesh Tewari, Alexander Johnson-Buck, Jieming Li, and Nils G. Walter
- Subjects
EGFR L858R ,Sensitivity and Specificity ,General Biochemistry, Genetics and Molecular Biology ,Article ,law.invention ,03 medical and health sciences ,law ,Molecule ,Humans ,Molecular Biology ,Polymerase chain reaction ,030304 developmental biology ,Fluorescent Dyes ,0303 health sciences ,Nonspecific binding ,Chemistry ,030302 biochemistry & molecular biology ,DNA ,Orders of magnitude (mass) ,Single Molecule Imaging ,ErbB Receptors ,MicroRNAs ,Genes ,Mutation ,Nucleic acid ,Biological system ,Nucleic acid detection - Abstract
Conventional methods for detecting small quantities of nucleic acids require amplification by the polymerase chain reaction (PCR), which necessitates prior purification and introduces copying errors. While amplification-free methods do not have these shortcomings, they are generally orders of magnitude less sensitive and specific than PCR-based methods. In this review, we provide a practical guide to a novel amplification-free method, single-molecule recognition through equilibrium Poisson sampling (SiMREPS), that provides both single-molecule sensitivity and single-bas e selectivity by monitoring the repetitive interactions of fluorescent probes to immobilized targets. We demonstrate how this kinetic fingerprinting filters out background arising from the inevitable nonspecific binding of probes, yielding virtually zero background signal. As practical applications of this digital detection methodology, we present the quantification of microRNA miR-16 and the detection of the mutation EGFR L858R with an apparent single-base discrimination factor of over 3 million.
- Published
- 2018
25. Exploring the speed limit of toehold exchange with a cartwheeling DNA acrobat
- Author
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Jieming, Li, Alexander, Johnson-Buck, Yuhe Renee, Yang, William M, Shih, Hao, Yan, and Nils G, Walter
- Subjects
Models, Molecular ,Kinetics ,Motion ,Fluorescence Resonance Energy Transfer ,Oligonucleotides ,DNA, Single-Stranded ,Nanotechnology ,Carbocyanines ,Fluorescent Dyes ,Nanostructures - Abstract
Dynamic DNA nanotechnology has yielded nontrivial autonomous behaviours such as stimulus-guided locomotion, computation and programmable molecular assembly. Despite these successes, DNA-based nanomachines suffer from slow kinetics, requiring several minutes or longer to carry out a handful of operations. Here, we pursue the speed limit of an important class of reactions in DNA nanotechnology-toehold exchange-through the single-molecule optimization of a novel class of DNA walker that undergoes cartwheeling movements over a field of complementary oligonucleotides. After optimizing this DNA 'acrobat' for rapid movement, we measure a stepping rate constant approaching 1 s
- Published
- 2017
26. Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm
- Author
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Neal W. Woodbury, Minghui Liu, Jinglin Fu, Yan Liu, Nils G. Walter, Alexander Johnson-Buck, Yuhe R. Yang, and Hao Yan
- Subjects
Enzyme complex ,Nanostructure ,Chemistry ,Biomedical Engineering ,Substrate (chemistry) ,Bioengineering ,Nanotechnology ,DNA ,Condensed Matter Physics ,Channelling ,Atomic and Molecular Physics, and Optics ,Nanostructures ,Enzyme catalysis ,Multienzyme Complexes ,Molecule ,General Materials Science ,A-DNA ,Electrical and Electronic Engineering ,Oxidoreductases ,Linker - Abstract
Swinging arms are a key functional component of multistep catalytic transformations in many naturally occurring multi-enzyme complexes. This arm is typically a prosthetic chemical group that is covalently attached to the enzyme complex via a flexible linker, allowing the direct transfer of substrate molecules between multiple active sites within the complex. Mimicking this method of substrate channelling outside the cellular environment requires precise control over the spatial parameters of the individual components within the assembled complex. DNA nanostructures can be used to organize functional molecules with nanoscale precision and can also provide nanomechanical control. Until now, protein-DNA assemblies have been used to organize cascades of enzymatic reactions by controlling the relative distance and orientation of enzymatic components or by facilitating the interface between enzymes/cofactors and electrode surfaces. Here, we show that a DNA nanostructure can be used to create a multi-enzyme complex in which an artificial swinging arm facilitates hydride transfer between two coupled dehydrogenases. By exploiting the programmability of DNA nanostructures, key parameters including position, stoichiometry and inter-enzyme distance can be manipulated for optimal activity.
- Published
- 2014
27. Discovering anomalous hybridization kinetics on DNA nanostructures using single-molecule fluorescence microscopy
- Author
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Nils G. Walter and Alexander Johnson-Buck
- Subjects
education.field_of_study ,Materials science ,Nanostructure ,Population ,Immobilized Nucleic Acids ,DNA, Single-Stranded ,Nanotechnology ,Single-molecule experiment ,General Biochemistry, Genetics and Molecular Biology ,Nanostructures ,Kinetics ,Förster resonance energy transfer ,Microscopy, Fluorescence ,DNA nanotechnology ,Microscopy ,Fluorescence Resonance Energy Transfer ,Fluorescence microscope ,DNA origami ,education ,Molecular Biology ,Fluorescent Dyes - Abstract
DNA nanostructures are finding diverse applications as scaffolds for molecular organization. In general, components such as nucleic acids, proteins, and nanoparticles are attached to addressable DNA nanostructures via hybridization, and there is interest in exploiting hybridization for localized computation on DNA nanostructures. This report details two fluorescence microscopy methods, single-particle fluorescence resonance energy transfer (spFRET) and DNA-PAINT (points accumulation for imaging in nanoscale topography), that have been successfully used to detect anomalies of hybridization reactions on individual DNA nanostructures. We compare and contrast the two techniques, highlighting their respective strengths in studying equilibrium and non-equilibrium hybridization as well as assessing the variability of behaviors within individual nanostructures and across a population of nanostructures.
- Published
- 2014
28. Beyond DNA origami: the unfolding prospects of nucleic acid nanotechnology
- Author
-
Alexander Johnson-Buck, Anthony J. Manzo, Nils G. Walter, and Nicole Michelotti
- Subjects
chemistry.chemical_compound ,Molecular recognition ,Materials science ,chemistry ,Nanoelectronics ,Biomedical Engineering ,Nucleic acid ,Medicine (miscellaneous) ,Nanomedicine ,DNA origami ,Bioengineering ,Nanotechnology ,DNA - Abstract
Nucleic acid nanotechnology exploits the programmable molecular recognition properties of natural and synthetic nucleic acids to assemble structures with nanometer-scale precision. In 2006, DNA origami transformed the field by providing a versatile platform for self-assembly of arbitrary shapes from one long DNA strand held in place by hundreds of short, site-specific (spatially addressable) DNA 'staples'. This revolutionary approach has led to the creation of a multitude of two-dimensional and three-dimensional scaffolds that form the basis for functional nanodevices. Not limited to nucleic acids, these nanodevices can incorporate other structural and functional materials, such as proteins and nanoparticles, making them broadly useful for current and future applications in emerging fields such as nanomedicine, nanoelectronics, and alternative energy.
- Published
- 2011
29. Kinetic fingerprinting to identify and count single nucleic acids
- Author
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Meiping Zhao, Nils G. Walter, María Dolores Giráldez, Xin Su, Alexander Johnson-Buck, and Muneesh Tewari
- Subjects
Genetics ,Extramural ,Sequence Analysis, RNA ,Biomedical Engineering ,Bioengineering ,Single-nucleotide polymorphism ,Computational biology ,Biology ,Applied Microbiology and Biotechnology ,Article ,MicroRNAs ,Limit of Detection ,False positive paradox ,Nucleic acid ,Molecular Medicine ,Diagnostic biomarker ,Humans ,Biotechnology ,Fluorescent Dyes - Abstract
MicroRNAs (miRNAs) have emerged as promising diagnostic biomarkers. We introduce a kinetic fingerprinting approach called Single Molecule Recognition through Equilibrium Poisson Sampling (SiMREPS) for the amplification-free counting of single unlabeled miRNA molecules, which circumvents thermodynamic limits of specificity and virtually eliminates false positives. We demonstrate high-confidence single-molecule detection of synthetic and endogenous miRNAs in both buffer and minimally treated biological liquids, as well as >500-fold discrimination between single nucleotide polymorphisms.
- Published
- 2015
30. DNA-cholesterol barges as programmable membrane-exploring agents
- Author
-
Hao Yan, Nils G. Walter, Shuoxing Jiang, and Alexander Johnson-Buck
- Subjects
Materials science ,Lipid Bilayers ,Oligonucleotides ,General Physics and Astronomy ,DNA, Single-Stranded ,Nanotechnology ,Polyethylene Glycols ,Diffusion ,chemistry.chemical_compound ,DNA nanotechnology ,Fluorescence microscope ,DNA origami ,General Materials Science ,Lipid bilayer ,Cytoskeleton ,Binding Sites ,Oligonucleotide ,Bilayer ,General Engineering ,Membranes, Artificial ,DNA ,Lipids ,Nanostructures ,Kinetics ,Membrane ,Cholesterol ,chemistry ,Microscopy, Fluorescence - Abstract
DNA nanotechnology enables the precise construction of nanoscale devices that mimic aspects of natural biomolecular systems yet exhibit robustly programmable behavior. While many important biological processes involve dynamic interactions between components associated with phospholipid membranes, little progress has been made toward creating synthetic mimics of such interfacial systems. We report the assembly and characterization of cholesterol-labeled DNA origami "barges" capable of reversible association with and lateral diffusion on supported lipid bilayers. Using single-particle fluorescence microscopy, we show that these DNA barges rapidly and stably embed in lipid bilayers and exhibit Brownian diffusion in a manner dependent on both cholesterol labeling and bilayer composition. Tracking of individual barges rapidly generates super-resolution maps of the contiguous regions of a membrane. Addition of appropriate command oligonucleotides enables membrane-associated barges to reversibly exchange fluorescent cargo with bulk solution, dissociate from the membrane, or form oligomers within the membrane, opening up new possibilities for programmable membrane-bound molecular devices.
- Published
- 2014
31. Multifactorial modulation of binding and dissociation kinetics on two-dimensional DNA nanostructures
- Author
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Shuoxing Jiang, Jeanette Nangreave, Alexander Johnson-Buck, Hao Yan, and Nils G. Walter
- Subjects
Oligonucleotide ,Mechanical Engineering ,Kinetics ,Bioengineering ,Nanotechnology ,General Chemistry ,DNA ,Condensed Matter Physics ,Dissociation (chemistry) ,Nanostructures ,chemistry.chemical_compound ,Förster resonance energy transfer ,chemistry ,DNA nanotechnology ,Biophysics ,DNA origami ,Nucleic Acid Conformation ,General Materials Science ,DNA microarray - Abstract
We use single-particle fluorescence resonance energy transfer (FRET) to show that organizing oligonucleotide probes into patterned two-dimensional arrays on DNA origami nanopegboards significantly alters the kinetics and thermodynamics of their hybridization with complementary targets in solution. By systematically varying the spacing of probes, we demonstrate that the rate of dissociation of a target is reduced by an order of magnitude in the densest probe arrays. The rate of target binding is reduced less dramatically, but to a greater extent than reported previously for one-dimensional probe arrays. By additionally varying target sequence and buffer composition, we provide evidence for two distinct mechanisms for the markedly slowed dissociation: direct hopping of targets between adjacent sequence-matched probes and nonsequence-specific, salt-bridged, and thus attractive electrostatic interactions with the DNA origami pegboard. This kinetic behavior varies little between individual copies of a given array design and will have significant impact on hybridization measurements and overall performance of DNA nanodevices as well as microarrays.
- Published
- 2013
32. Super-Resolution Fingerprinting Detects Chemical Reactions and Idiosyncrasies of Single DNA Pegboards
- Author
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Hao Yan, Jeanette Nangreave, Nils G. Walter, Do-Nyun Kim, Mark Bathe, Alexander Johnson-Buck, Massachusetts Institute of Technology. Department of Biological Engineering, Bathe, Mark, and Kim, Do-Nyun
- Subjects
Chemical imaging ,Materials science ,Oligonucleotide ,Mechanical Engineering ,Bioengineering ,Nanotechnology ,DNA ,General Chemistry ,Condensed Matter Physics ,DNA Fingerprinting ,Superresolution ,Fluorescence ,Article ,chemistry.chemical_compound ,chemistry ,Feature (computer vision) ,DNA origami ,General Materials Science ,DNA Probes ,Nanoscopic scale - Abstract
We employ the single-particle fluorescence nanoscopy technique points accumulation for imaging in nanoscale topography (PAINT) using site-specific DNA probes to acquire two-dimensional density maps of specific features patterned on nanoscale DNA origami pegboards. We show that PAINT has a localization accuracy of ∼10 nm that is sufficient to reliably distinguish dense (>10[superscript 4] features μm[superscript –2]) sub-100 nm patterns of oligonucleotide features. We employ two-color PAINT to follow enzyme-catalyzed modification of features on individual origami and to show that single nanopegboards exhibit stable, spatially heterogeneous probe-binding patterns, or “fingerprints.” Finally, we present experimental and modeling evidence suggesting that these fingerprints may arise from feature spacing variations that locally modulate the probe binding kinetics. Our study highlights the power of fluorescence nanoscopy to perform quality control on individual soft nanodevices that interact with and position reagents in solution., National Science Foundation (U.S.) (Collaborative Research Award CCF-0829579), United States. Multidisciplinary University Research Initiative (W911NF-12-1-0420)
- Published
- 2013
33. Metal ions: supporting actors in the playbook of small ribozymes
- Author
-
Sarah E. McDowell, Alexander Johnson-Buck, and Nils G. Walter
- Subjects
Regulation of gene expression ,Ions ,Models, Molecular ,Small RNA ,Molecular Structure ,RNase P ,Ribozyme ,RNA ,Group II intron ,Biology ,Catalysis ,Article ,Biochemistry ,Metals ,Catalytic Domain ,biology.protein ,Nucleic Acid Conformation ,RNA, Catalytic ,Cellular metal ion homeostasis ,Ligase ribozyme - Abstract
Since the 1980s, several small RNA motifs capable of chemical catalysis have been discovered. These small ribozymes, composed of between approximately 40 and 200 nucleotides, have been found to play vital roles in the replication of subviral and viral pathogens, as well as in gene regulation in prokaryotes, and have recently been discovered in noncoding eukaryotic RNAs. All of the known natural small ribozymes – the hairpin, hammerhead, hepatitis delta virus, Varkud satellite, and glmS ribozymes – catalyze the same self-cleavage reaction as RNase A, resulting in two products, one bearing a 2′-3′ cyclic phosphate and the other a 5′-hydroxyl group. Although originally thought to be obligate metalloenzymes like the group I and II self-splicing introns, the small ribozymes are now known to support catalysis in a wide variety of cations that appear to be only indirectly involved in catalysis. Nevertheless, under physiologic conditions, metal ions are essential for the proper folding and function of the small ribozymes, the most effective of these being magnesium. Metal ions contribute to catalysis in the small ribozymes primarily by stabilizing the catalytically active conformation, but in some cases also by activating RNA functional groups for catalysis, directly participating in catalytic acid-base chemistry, and perhaps by neutralizing the developing negative charge of the transition state. Although interactions between the small ribozymes and cations are relatively nonspecific, ribozyme activity is quite sensitive to the types and concentrations of metal ions present in solution, suggesting a close evolutionary relationship between cellular metal ion homeostasis and cation requirements of catalytic RNAs, and perhaps RNA in general.
- Published
- 2011
34. A Bird's Eye View
- Author
-
Chamaree de Silva, Nicole Michelotti, Nils G. Walter, Anthony J. Manzo, and Alexander Johnson-Buck
- Subjects
Fluorescence-lifetime imaging microscopy ,Materials science ,Scale (ratio) ,Nano ,DNA nanotechnology ,Molecular motion ,Nanotechnology ,Nanometre ,Tracking (particle physics) ,Characterization (materials science) - Abstract
Recent improvements in methods of single-particle fluorescence tracking have permitted detailed studies of molecular motion on the nanometer scale. In a quest to introduce these tools to the burgeoning field of DNA nanotechnology, we have exploited fluorescence imaging with one-nanometer accuracy (FIONA) and single-molecule high-resolution colocalization (SHREC) to monitor the diffusive behavior of synthetic molecular walkers, dubbed "spiders," at the single-molecule level. Here we discuss the imaging methods used, results from tracking individual spiders on pseudo-one-dimensional surfaces, and some of the unique experimental challenges presented by the low velocities (approximately 3 nm/min) of these nanowalkers. These experiments demonstrate the promise of fluorescent particle tracking as a tool for the detailed characterization of synthetic molecular nanosystems at the single-molecule level.
- Published
- 2010
35. The shape-shifting quasispecies of RNA: one sequence, many functional folds
- Author
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Alexander Johnson-Buck, Nils G. Walter, and Matthew S. Marek
- Subjects
Protein Folding ,Sarcina ,Population ,General Physics and Astronomy ,Ricin ,Computational biology ,Ribosome ,Article ,RNA, Catalytic ,Physical and Theoretical Chemistry ,Nucleic acid structure ,education ,education.field_of_study ,Base Sequence ,biology ,Chemistry ,Ribozyme ,Intron ,Proteins ,RNA ,Aptamers, Nucleotide ,Biochemistry ,biology.protein ,Nucleic Acid Conformation ,Protein folding ,Hairpin ribozyme ,Ribosomes - Abstract
E Unus pluribum, or "Of One, Many", may be at the root of decoding the RNA sequence-structure-function relationship. RNAs embody the large majority of genes in higher eukaryotes and fold in a sequence-directed fashion into three-dimensional structures that perform functions conserved across all cellular life forms, ranging from regulating to executing gene expression. While it is the most important determinant of the RNA structure, the nucleotide sequence is generally not sufficient to specify a unique set of secondary and tertiary interactions due to the highly frustrated nature of RNA folding. This frustration results in folding heterogeneity, a common phenomenon wherein a chemically homogeneous population of RNA molecules folds into multiple stable structures. Often, these alternative conformations constitute misfolds, lacking the biological activity of the natively folded RNA. Intriguingly, a number of RNAs have recently been described as capable of adopting multiple distinct conformations that all perform, or contribute to, the same function. Characteristically, these conformations interconvert slowly on the experimental timescale, suggesting that they should be regarded as distinct native states. We discuss how rugged folding free energy landscapes give rise to multiple native states in the Tetrahymena Group I intron ribozyme, hairpin ribozyme, sarcin-ricin loop, ribosome, and an in vitro selected aptamer. We further describe the varying degrees to which folding heterogeneity impacts function in these RNAs, and compare and contrast this impact with that of heterogeneities found in protein folding. Embracing that one sequence can give rise to multiple native folds, we hypothesize that this phenomenon imparts adaptive advantages on any functionally evolving RNA quasispecies.
- Published
- 2011
36. Imaging an Expanding Molecular Robot World Using Super-Accuracy Single-Molecule Fluorescence Microscopy
- Author
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L. Devon Triplett, Milan N. Stojanovic, Jeanette Nangreave, Steven Taylor, Nils G. Walter, Alexander Johnson-Buck, Anthony J. Manzo, Nicole Michelotti, and Hao Yan
- Subjects
Streptavidin ,Spider ,chemistry.chemical_compound ,Fluorophore ,Total internal reflection fluorescence microscope ,chemistry ,Biophysics ,Robot ,Nanotechnology ,A-DNA ,Biological system ,Single-molecule experiment ,Random walk - Abstract
We recently demonstrated the concept of molecular robotics with a synthetic DNA-based nanowalker, dubbed a “spider” composed of a streptavidin protein “body” attached to three biotinylated DNA enzyme legs, along a one-dimensional track of chimeric DNA-RNA substrates positioned on a DNA origami (1,2). By cleaving its substrates, the spider weakens the binding energy between its legs and previously visited sites, resulting in a biased, processive, random walk towards fresh substrate. Additional components are now being incorporated into the spider world to increase its versatility and complexity in behavior. For example, we are implementing a second spider that walks on a different substrate. This spider can be differentially controlled from our original nanowalker via changes in buffer conditions that favor one spider over the other and vice versa. This advance will allow us to devise a controlled “spider race” towards a common goal post; depending on the predetermined winner, a payload will either be released from the goal or not, behaving as an XOR gate. By fluorophore labeling the different components, we aim to characterize this spider race at the single molecule level using super-accuracy total internal reflection fluorescence microscopy (TIRFM).1. Lund, K., Manzo, A.J., Dabby, N., Michelotti, N., Johnson-Buck, A., Nangreave, J., Taylor, S., Pei, R., Stojanovic, M.N., Walter, N.G., Winfree, E., and Yan, H. (2010) Molecular robots guided by prescriptive landscapes. Nature 465, pp. 206–210.2. Michelotti, N., de Silva, C., Johnson-Buck, A.E., Manzo, A.J., Walter, N.G. (2010) A bird's eye view: tracking slow nanometer-scale movements of single molecular nano-assemblies. Methods Enzymol. 475, pp. 121–148.
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