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Single-molecule chemical reactions on DNA origami

General Scheme. Some Biotinylated Chemical Moiety. Index of DNA Dumbells. Biotin labels. Addition of Streptavidin. Origami Schematic. Method. Conjugate different functional groups to selected staple strandsBind biotin to selected staple strandsVisualize formation or cleavage of individual chemical bonds by attachment or removal of biotin-streptavidin complex12 specific staples modifiedImaged under liquid in tapping mode AFM.

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Single-molecule chemical reactions on DNA origami

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    1. “Single-molecule chemical reactions on DNA origami” Niels V. Voigt, Thomas Tørring, AlexandruRotaru, Mikkel F. Jacobsen, Jens B. Ravnsbæk, RameshSubramani, WaelMamdouh, JørgenKjems, AndriyMokhir, FlemmingBesenbacher& Kurt VesteragerGothelf, Nat Nano 5, no. 3 (March 2010): 200-203.

    3. Origami Schematic

    4. Method Conjugate different functional groups to selected staple strands Bind biotin to selected staple strands Visualize formation or cleavage of individual chemical bonds by attachment or removal of biotin-streptavidin complex 12 specific staples modified Imaged under liquid in tapping mode AFM

    5. Linkers Linker A – non-cleavable linker Linker B – disulphide bond cleaved by reduction Applied 1,4-dithiothreitol (DTT) Linker C – 1,2-bis(alkylthio)ethene cleaved by singlet oxygen Applied singlet oxygen photosensitizer eosin and white light

    6. Linker Images

    7. Chemical Bond CleavageRxns Assembly Anneal Concentration: 5nM 1:30 viral DNA:staples Annealed in 1 x TAE-Mg2+ (12.5mM Mg Acetate) – pH=8.0 Thermal Anneal: 90°C - 4°C over 14 hrs (0.1°C/min) Purified using 100kDa MWCO centrifugal filter Streptavidin (2uM) incubated for 2 hrs

    8. Reductive Cleavage with DTT In solution 10mM DTT incubated with unfiltered origamis for 6 hrs at RT Streptavidin added for 2 hrs at RT AFM On mica Filtered origami added to cleaved mica (adsorbed 3 min) Incubate with 30mM DTT 4 hrs at RT Washed surface with 1xTAE-Mg2+ Results were comparable

    9. Cleavage with singlet Oxygen On mica Sample deposited on mica after DTT treatment Added eosin (100uM) and DTT (1mM) Irradiate samples with 150 W for 60 min Remove buffer and wash 2x with 1xTAE-Mg2+

    10. Single Molecule Bond Formation Alkyne Copper(I)-catalyzed click reaction w/ azide to form triazole Amine Reacts w/ N-hydroxysuccinimide activates ester (NHS-ester) to form amide Azide Copper(I)-catalyzed click reaction w/ alkyne to form triazole

    11. Single Molecule Images

    12. Chemical Bond FormingRxns Assembly Anneal Concentration: 10 nM 1:10 viral DNA:staples Annealed in 20mM HEPES buffer (12.5mM MgCl2) – pH=7.4 Thermal Anneal: 90°C - 4°C over 14 hrs (0.1°C/min)

    13. SM reaction Conditions Origami deposited on Mica under HEPES/Mg2+ buffer (pH=7.4) Alkyne Copper(I)-THTA in DMF/buffer mixture Amine Slightly alkaline buffer/DMF mixture (pH=8.3) Azide Copper(I)-THTA catalyst Incubate reagents for 20 min Incubate with streptavidin for 5 min

    14. First Click Rxn On mica Origami added to cleaved mica (adsorbed 3 min) Add HEPES/Mg2+ buffer Image with AFM Remove buffer and add coupling solution Coupling soln: CuSO4 (200uM), THTA(1.4mM), Sodium ascorbate (20mM), azidefunctionalized biotin (10mM) Incubate for 20 min Wash with HEPES/Mg2+ buffer Incubate with streptavidin (200nM) in HEPES/Mg2+ buffer 5 min Wash and Image with AFM

    15. Amine and Activated Acid Rxn On mica Origami added to cleaved mica (adsorbed 3 min) Add HEPES/Mg2+ buffer Image with AFM Remove buffer and add coupling solution Coupling soln: Biotin-NHS (10mM), 1:4 DMF/Buffer (HEPES; 20mM, Mg2+; 12.5mM, pH=8.26) Incubate for 20 min Wash with HEPES/Mg2+ buffer Incubate with streptavidin (200nM) in HEPES/Mg2+ buffer 5 min Wash and Image with AFM

    16. Second Click Rxn On mica Origami added to cleaved mica (adsorbed 3 min) Add HEPES/Mg2+ buffer Image with AFM Remove buffer and add coupling solution Coupling soln: CuSO4 (200uM), THTA(1.4mM), Sodium ascorbate (20mM), alkyne functionalized biotin (5mM in DMF/HEPES/Mg2+) Incubate for 20 min Wash with HEPES/Mg2+ buffer Incubate with streptavidin (200nM) in HEPES/Mg2+ buffer 5 min Wash and Image with AFM Note: Imaging conditions make index difficult to see

    17. Yield Calculation

    18. Yields

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