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PreprintCited by 5Force Microscopy Techniques and ApplicationsAdvanced biosensing and bioanalysis techniquesAdvanced Fluorescence Microscopy Techniques

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Mapping Single-molecule Protein Complexes in 3D with DNA Nanoswitch Calipers

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Shrestha, Prakash, Yang, Darren, Shih, William M., Wong, Wesley P.·2023·Journal of the American Chemical Society
DOI 10.1101/2023.07.10.548386PMC PMC10755700

Preprint — published as 10.1021/jacs.3c10262

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Abstract

The ability to accurately map the 3D geometry of single-molecule complexes in trace samples would lead to new insights into molecular mechanics and provide an approach for single-molecule structural proteomics. To enable this, we have developed a high-resolution force-spectroscopy method capable of measuring multiple distances between labeled sites in natively folded protein complexes. Our approach combines reconfigurable nanoscale devices we call DNA Nanoswitch Calipers, which we have previously introduced, with a force-based barcoding system to distinguish each measurement location. We demonstrate our approach by reconstructing the tetrahedral geometry of biotin-binding sites in natively folded streptavidin, with 1.5-2.5 Å agreement to previously reported structures.

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Figure 1 Overview of single-molecule 3D mapping with DNA nanoswitch calipers (DNC). (Left) Natively folded biomolecular complex labeled with ssDNA handles: a strong handle (purple) that serves as an anchoring site for DNC and shorter weak handles (red). (Middle) Multiple distances are measured between red and purple handles using a DNC actuated by mechanical force. Each DNC has one “grabber” complementary to the weak handles and one complementary to the strong handle. As force is cycled between low and high forces, the caliper alternates between looped and linear states, due to engagement then release of the weak handle. (Top right) Distribution of measured distances d calculated by subtracting the change in length ΔL from the effective loop length L0, which serves as a reference to convert changes in length to absolute distance. (Bottom right) Reconstructed geometry for positions of DNA-labeled sites in the folded biomolecular complex.
Figure 1 Overview of single-molecule 3D mapping with DNA nanoswitch calipers (DNC). (Left) Natively folded biomolecular complex labeled with ssDNA handles: a strong handle (purple) that serves as an anchoring site for DNC and shorter weak handles (red). (Middle) Multiple distances are measured between red and purple handles using a DNC actuated by mechanical force. Each DNC has one “grabber” complementary to the weak handles and one complementary to the strong handle. As force is cycled between low and high forces, the caliper alternates between looped and linear states, due to engagement then release of the weak handle. (Top right) Distribution of measured distances d calculated by subtracting the change in length ΔL from the effective loop length L0, which serves as a reference to convert changes in length to absolute distance. (Bottom right) Reconstructed geometry for positions of DNA-labeled sites in the folded biomolecular complex.CC BY-NC-ND · PubMed Central
Figure 2 Single-molecule distance measurements of streptavidin–biotin complexes with only two biotin sites occupied. (A) Structure of streptavidin–biotin complex (PDB entry 6M9B) (left) with a schematic representation (right) where spheres indicate biotins bound to streptavidin. (B) Schematic of streptavidin bound to two biotinylated ssDNA handles illustrating three distinct configurations. The longer (purple) strong handle provides strong attachment to the DNC, while the shorter (red) weak handle shears off at lower force. Biotinylated handles are hybridized to the DNC construct prior to the introduction of streptavidin to yield one pair of handles per molecule (Figure S1). (C) DNC tethered between two optically trapped microbeads and actuated by force to measure distances between biotin-binding sites in streptavidin. DNC looping occurs when both handles are engaged; unlooping occurs when the weak handle shears off at high force; relooping occurs with weak handle rebinding at low force. (D) Data showing DNC extension over cycles of low and high force (raw data in black, 200-point sliding window average in red). (E) Zoom-in of blue rectangle in D, showing change in extension due to DNC unlooping. (F) Distance histogram calculated by subtracting ΔL from effective loop length (L0). (G) Histogram of per-molecule average distances for 27 molecular complexes. Plus–minus values indicate sigma fitting parameters from the multipeak Gaussian fit. (H) DNC determined positions of carbon in the carboxylic group of each biotin (black spheres) superimposed on the crystal structure (PDB 6M9B).
Figure 2 Single-molecule distance measurements of streptavidin–biotin complexes with only two biotin sites occupied. (A) Structure of streptavidin–biotin complex (PDB entry 6M9B) (left) with a schematic representation (right) where spheres indicate biotins bound to streptavidin. (B) Schematic of streptavidin bound to two biotinylated ssDNA handles illustrating three distinct configurations. The longer (purple) strong handle provides strong attachment to the DNC, while the shorter (red) weak handle shears off at lower force. Biotinylated handles are hybridized to the DNC construct prior to the introduction of streptavidin to yield one pair of handles per molecule (Figure S1). (C) DNC tethered between two optically trapped microbeads and actuated by force to measure distances between biotin-binding sites in streptavidin. DNC looping occurs when both handles are engaged; unlooping occurs when the weak handle shears off at high force; relooping occurs with weak handle rebinding at low force. (D) Data showing DNC extension over cycles of low and high force (raw data in black, 200-point sliding window average in red). (E) Zoom-in of blue rectangle in D, showing change in extension due to DNC unlooping. (F) Distance histogram calculated by subtracting ΔL from effective loop length (L0). (G) Histogram of per-molecule average distances for 27 molecular complexes. Plus–minus values indicate sigma fitting parameters from the multipeak Gaussian fit. (H) DNC determined positions of carbon in the carboxylic group of each biotin (black spheres) superimposed on the crystal structure (PDB 6M9B).CC BY-NC-ND · PubMed Central
Figure 3 3D mapping of streptavidin–biotin complex with force-based barcodes to resolve multiple distance measurements per molecule. (A) Schematic showing incorporation and readout of hairpin barcodes incorporated into weak handles (red). First, hairpin opening enables handle identification from the change in length, and then loop opening at a higher force enables the distance between handles to be measured as before. Dropping the force enables rebinding to another handle. (B) Outline of distance measurement strategy consisting of a force ramp for hairpin readout, followed by jumps in force to ∼20 pN to measure the distance, ∼30 pN to shear the weak handle, ∼20 pN to measure the change in length, and then ∼0 pN to enable rebinding. Three cycles are shown, illustrating unfolding signatures of the three barcodes. (C) Extension vs time data for a DNC over multiple force cycles (raw data in gray, smoothed in red). (D) Zoom-in of a section (indicated by purple arrows) from C depicting the sequential readout of all three barcodes. (E) Force vs extension curves of repeated cycles depicting the measurement of all three barcode lengths in one streptavidin molecule. (F) Scatter plot of measured barcode lengths vs handle distances in a single molecule. For each cluster, red circles indicate mean, and error bars indicate standard deviation. (G) Histogram of per-molecule distances aggregated over multiple molecules. Plus–minus values indicate sigma fitting parameters from the multipeak Gaussian fit. Blue vertical lines indicate expected distances between biotin-binding sites based on the crystal structure (PDB 6M9B). (H) Superposition of the DNC determined the position of the carbon in the carboxylic group of each biotin (black spheres) on the crystal structure.
Figure 3 3D mapping of streptavidin–biotin complex with force-based barcodes to resolve multiple distance measurements per molecule. (A) Schematic showing incorporation and readout of hairpin barcodes incorporated into weak handles (red). First, hairpin opening enables handle identification from the change in length, and then loop opening at a higher force enables the distance between handles to be measured as before. Dropping the force enables rebinding to another handle. (B) Outline of distance measurement strategy consisting of a force ramp for hairpin readout, followed by jumps in force to ∼20 pN to measure the distance, ∼30 pN to shear the weak handle, ∼20 pN to measure the change in length, and then ∼0 pN to enable rebinding. Three cycles are shown, illustrating unfolding signatures of the three barcodes. (C) Extension vs time data for a DNC over multiple force cycles (raw data in gray, smoothed in red). (D) Zoom-in of a section (indicated by purple arrows) from C depicting the sequential readout of all three barcodes. (E) Force vs extension curves of repeated cycles depicting the measurement of all three barcode lengths in one streptavidin molecule. (F) Scatter plot of measured barcode lengths vs handle distances in a single molecule. For each cluster, red circles indicate mean, and error bars indicate standard deviation. (G) Histogram of per-molecule distances aggregated over multiple molecules. Plus–minus values indicate sigma fitting parameters from the multipeak Gaussian fit. Blue vertical lines indicate expected distances between biotin-binding sites based on the crystal structure (PDB 6M9B). (H) Superposition of the DNC determined the position of the carbon in the carboxylic group of each biotin (black spheres) on the crystal structure.CC BY-NC-ND · PubMed Central

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Citations

Cites

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Metadata

Authors
Shrestha, P., Yang, D., Shih, W. M., & Wong, W. P.
Affiliations
  • Boston Children's Hospital (US)
  • Harvard University (US)
Corresponding author
Wong, Wesley P.
ORCID
Venue
Journal of the American Chemical Society
Publisher
openRxiv
Published
2023-07-11
Volume
145
Issue
51
Pages
27916-27921
ISSN
0002-7863 (ISSN-L)
DOI
10.1101/2023.07.10.548386
PMC
PMC10755700
OpenAlex
W4389764840
Cited by
5
References
1
Citations by year
2026: 1 · 2025: 2 · 2024: 2
FWCI
0.58
Type
journal article
Language
en
Open access
Licence
cc-by-nc-nd
Locations
  • Journal of the American Chemical Society (publishedVersion, cc-by-nc-nd)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion)
Funders
  • Alfred P. Sloan Foundation (G-2021-169145)
  • Harvard University
  • National Institutes of Health
  • Hansjörg Wyss Institute for Biologically Inspired Engineering, Harvard University
  • Office of Naval Research (N00014-15-1-0073)
  • National Institute of General Medical Sciences (GM119537, R35GM119537)
Topics
  • Physical Sciences › Physics and Astronomy › Atomic and Molecular Physics, and Optics › Force Microscopy Techniques and Applications
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Advanced biosensing and bioanalysis techniques
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
Keywords
single-molecule force spectroscopy, streptavidin, biotin binding site, tetrahedral geometry, nanodevices

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