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 Nano Scale Structures

2

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

Array Applications

-Attach any biological molecules to the origami -Spatially resolvable grid for taking single molecule measurements in bulk -observe known quantity of molecules

2 microns

10 microns

Our grid

• small chip containing thousands of DNA origami

400nM X 400nM

7uM x 1uM

10 microns

5

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

Image processing

7

Contrast Enhanced Raw Image

Local Intensity Threshold Spot Areas

Spot Intensity = Pixel Intensity – Average Background

Spot Intensity

1 2 3 4 5 6 7 8 9 10

Average spot intensity

Ideal histogram -Read out intensity of each spot and subtract background intensity -Use intensity to count the number of fluorophores

8

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

9

Photo bleaching

-Observed dark states where fluorophore temporarily cannot emit light

-Fluorphore is oxidized and Becomes permanently dark 10

Time Time

Inte

nsi

ty

Inte

nsi

ty

Estimated

# of Flu

oro

ph

ores

5

4

3

2

1

0

photo bleaching

-Observed exponential decay curve for spot intensity -Time constant of ~200 seconds under oil

632 Spots 82 Spots

11

Exposure Time

Inte

nsi

ty

400nm grid -1 observed intensity Peak -Intensity blending between adjacent fluorophores -difficult to detect local background noise

12 Intensity

# o

f O

bse

rvat

ion

s

1x7 micron grid -Poor grid alignment -Roughly 225 spots on grid out of estimated 858 – 26% filled -632 spots total with many origami at each location -multi peak histogram can distinguish absence of fluorophores

13

New Histogram Results

40

# o

f O

bse

rvat

ion

s 250

0 0

- High certainty of 3 evenly spaced bins in 150 spot segment

Intensity (AU) 500 0 1000 0 3200 1600

Intensity (AU)

-600 spots reveals 4+ peaks but also more uncertainty due to noise variation

Going forward Ideal histogram Current Histogram

15

- Code improvements - Improved sample quality - Imaging technique

2 microns 10 microns

Correspond AFM to Fluorescence

Silver DNA Nanoclusters

• Need to distinguish the fluorescent products of Silver DNA synthesis

• Attach Silver DNA instead of Dye

• Check chemical yields without purification

16

Acknowledgments

Deborah Fygenson

Travis Del Bonis-O’Donnel

Ashwin Gopinath

Deborah Clayton-Warwick

The Worster Family

17