Attaching Fluorescent Nanoclusters to DNA Origami Microarrays John - - PowerPoint PPT Presentation

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Attaching Fluorescent Nanoclusters to DNA Origami Microarrays John - - PowerPoint PPT Presentation

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Attaching Fluorescent Nanoclusters to DNA Origami Microarrays John Devany 10/31/14 Worster Fellowship Presentation 1 DNA Origami -Long piece of DNA facilitated to fold by smaller DNA staple Strands -Create self assembling 2 Dimensional

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SLIDE 1

Attaching Fluorescent Nanoclusters to DNA Origami Microarrays

John Devany 10/31/14 Worster Fellowship Presentation

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SLIDE 2

DNA Origami

  • Long piece of DNA facilitated to fold by smaller

DNA staple Strands

  • Create self assembling 2 Dimensional Nano Scale

Structures 2

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SLIDE 3

DNA Origami Microarrays

  • 130nm equilateral Triangle
  • 3 dye molecules
  • Inner strands charged to

couple with grid space

  • Negatively charged

areas on surface of grid

  • origami bind to grid

locations

  • covalent bonding

holds origami in place 3

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SLIDE 4

Array Applications

  • Attach any biological molecules to the
  • rigami
  • Spatially resolvable grid for taking

single molecule measurements in bulk

  • observe known quantity of molecules
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SLIDE 5

2 microns 10 microns

Our grid

  • small chip containing thousands of DNA origami

400nM X 400nM 7uM x 1uM

10 microns

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SLIDE 6

Goals

# of Fluorophores 4+ 3+ 1-2 0 # of Origami per location 0 1 2

Identify

Image Processing

Ideal histogram

6 0 1 2 3 4 5

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SLIDE 7

Image processing

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Contrast Enhanced Raw Image Local Intensity Threshold Spot Areas

Spot Intensity = Pixel Intensity – Average Background

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SLIDE 8

Spot Intensity

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Average spot intensity Ideal histogram

  • Read out intensity of each spot and

subtract background intensity

  • Use intensity to count the number of

fluorophores 8

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SLIDE 9

Grid Detection

  • Plot intensity across columns and rows
  • peaks indicate spot locations
  • generate grid based on spot locations
  • determine fill fraction from number of

empty grid locations

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SLIDE 10

Photo bleaching

  • Observed dark states where fluorophore

temporarily cannot emit light

  • Fluorphore is oxidized and

Becomes permanently dark 10

Time Time Intensity Intensity Estimated # of Fluorophores 5 4 3 2 1

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SLIDE 11

photo bleaching

  • Observed exponential decay curve for spot

intensity

  • Time constant of ~200 seconds under oil

632 Spots 82 Spots

11 Exposure Time Intensity

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SLIDE 12

400nm grid

  • 1 observed intensity Peak
  • Intensity blending between

adjacent fluorophores

  • difficult to detect local

background noise

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Intensity # of Observations

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SLIDE 13

1x7 micron grid

  • Poor grid alignment
  • Roughly 225 spots on grid out
  • f estimated 858 – 26% filled
  • 632 spots total with many origami at

each location

  • multi peak histogram can distinguish

absence of fluorophores

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SLIDE 14

New Histogram Results

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# of Observations

250

  • High certainty of 3 evenly spaced bins

in 150 spot segment

Intensity (AU)

500 1000 3200 1600

Intensity (AU)

  • 600 spots reveals 4+ peaks but also

more uncertainty due to noise variation

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SLIDE 15

Going forward

Ideal histogram Current Histogram

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  • Code improvements
  • Improved sample quality
  • Imaging technique

2 microns 10 microns

Correspond AFM to Fluorescence

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SLIDE 16

Silver DNA Nanoclusters

  • Need to distinguish

the fluorescent products of Silver DNA synthesis

  • Attach Silver DNA

instead of Dye

  • Check chemical yields

without purification

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SLIDE 17

Acknowledgments

Deborah Fygenson Travis Del Bonis-O’Donnel Ashwin Gopinath Deborah Clayton-Warwick The Worster Family

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