Bioresponsive quantum dot lattices for applications in biosensing and conditionally activated RNA interference

1. Bioresponsive Quantum Dot Lattices
for Applications in Biosensing
and Conditionally Activated RNA Interference
Morgan Chandler
Graduate Student, Nanoscale Science
Advisor: Dr. Kirill A. Afonin
Department of Chemistry, University of North Carolina at Charlotte

2. Quantum dots are semiconductor nanocrystals with
tunable light emission.
• Quantum dots (QDs) are increasingly used for applications
such as in solar cells, bioimaging, and LEDs
• Due to quantum confinement, their emission depends upon
their sizes, which are usually around 2-10 nm in diameter
• QDs exhibit intermittent photoluminescence known as
“blinking” in which they fluctuate between bright “on” and
dark “off” states

3. How can QD blinking be utilized as a detection system?
OFF OFF
ON ON ON ON ON
ON ON ON
A single blinking QD
results in two possible
states: ON or OFF.
When multiple blinking QDs are brought together, it is more likely that at least one in the detection
volume is in an ON state. Therefore, the emission appears continuous.

4. • Develop a method of assembling QDs to assess blinking versus quasicontinuous emission
• Design a strategy for making the assembly conditionally-activated only in the presence of a
biologically-relevant target molecule
Research Goals:
How can single QDs be brought together?
Roark B, Tan JA, Ivanina A, Chandler M, Castaneda J, Kim HS, Jawahar S, Viard M, Talic S, Wustholz KL, Yingling YG, Jones M, Afonin KA. ACS Sensors, 2016.

5. Research Methods:
DNA-driven QD assembly is driven by a target strand
• The target strand (4) is a fragment of oncogene
KRAS with a codon 12 mutation known to be
frequent in various cancers
• The sensor is composed of a DNA duplex (1 and 2)
and single DNA (3)
• In the presence of the target, the thermodynamically-
driven displacement of DNA strands (assisted by
single-stranded DNA toeholds) results in a double
biotinylated DNA duplex (2+3)
• The double biotinylated DNA duplex rapidly crosslinks
streptavidin-decorated QDs, resulting in the
formation of a lattice
• For this and all the following studies (except where
noted), QD545 (green) is used
(1)
(4)
(2)
(3)
(2+3)
(1+4)

6. Roark B, Tan JA, Ivanina A, Chandler M, Castaneda J, Kim HS, Jawahar S, Viard M, Talic S, Wustholz KL, Yingling YG, Jones M, Afonin KA. ACS Sensors, 2016.
Research Methods:
Fluorescence microscopy can be used to assess blinking
A single QD exhibits
streaking due to blinking
Assembled QDs exhibit
little streaking
Blinking traces from bright spots in fluorescence images
either (A) without assembly or (B) with assembly and their
corresponding intensity histograms (C and D)

7. Research Methods:
Gel electrophoresis can also be used to visualize lattices
• Agarose gels with ethidium bromide (EtBr) are useful
for quickly assessing lattice formation
• Free QDs do not migrate far on their own, but when
bound to DNA, the increase in negative charge results
in movement through the gel
• When QD/DNA lattices form, the large size prevents
migration out of the well
• Free DNA (such as the double-stranded DNA in the
third lane) travels the fastest; any excess DNA in the
assembly can be visualized
• Bovine serum albumin (BSA) is a component of the QD
buffer and is present in all samples containing QDs

8. Biotinylated DNAs crosslink QDs for lattice formation
2% agarose gel
2% agarose gel
Quantum Dots
BSA (in QD buffer)
DNA
BSA (in QD buffer)
DNA
To verify single-stranded biotinylated DNAs
bind QDs:
• Increasing amounts of DNA were added
to QDs
• Once bound, the greater negative
charge increased QD migration through
the gel
To determine the number of binding sites
per QD:
• Increasing amounts of the (2+3) double-
biotinylated DNA duplex were added
with QDs
• Lattice formation, as seen in the wells,
first occurs with approximately 10
duplexes per QD

9. Lattice formation is fast with a low limit of detection
2% agarose gel
2% agarose gel
To determine the limit of detection:
• Decreasing concentrations of crosslinked
QDs were visualized
• The lowest concentration of lattices required
for gel visualization is approximately 4.8 nM
To determine the speed of lattice assembly:
• Crosslinked QDs were frozen at various
timepoints from 0.5 to 15 minutes
• When loaded, all timepoints showed lattice
assembly, even at 0.5 minutes
• DNase digestion of lattices releases free QDs
BSA
BSA

10. QD lattice formation can be functionalized to trigger
RNA interference
• As an alternative design strategy,
QDs bound to hybrid DNA/RNA
duplexes can interact with
complementary hybrid DNA/RNA
duplexes bound to QDs
• The double-biotinylated DNA
duplexes push out double-
stranded Dicer Substrate (DS)
RNAs which then enter the RNA
interference (RNAi) pathway for
gene knockdown
• Assembly was verified by gel and
microscopy

11. QD lattices were shown to release DS RNAs in cells
• Cognate hybrid QDs (sense and antisense) carrying split
DS RNAs against green fluorescent protein (GFP) were
added to human breast cancer cells expressing
enhanced GFP (MDA-MB-231 eGFP)
• With only either hybrid QD, no silencing was observed
• Addition of both cognate hybrid QDs showed reduced
GFP expression

12. Intracellular co-localization of QD lattices
• Streptavidin-decorated QD605 bound to hybrid duplexes and
streptavidin-decorated QD545 bound to hybrid duplexes were
shown to assemble in human breast cancer cells
• Using confocal microscopy, the emission from each was
visualized and superpositioned to show the complete overlap,
of signals corresponding to QD605/QD545 lattice formation
Differential interference
contrast
QD605 emission QD545 emission Superposition of
previous three images

13. • Biotinylated DNAs can be utilized to program the assembly of streptavidin-decorated QDs
into lattices
• The quasicontinuous fluorescence of QDs can be used to detect the presence of a single
point mutation for rapid biosensing
• Lattice formation is fast with a low limit of detection
• Can be assessed using fluorescence microscopy and gel electrophoresis
• QD lattices can introduce therapeutic Dicer Substrate RNAs into cells for gene silencing
while their assembly in cells can be tracked
• Combining the release of DS RNA with the activation of a specific target sequence
• Building upon current platform with other potential target sequences and other
incorporated therapeutic targets
Conclusions
Future Directions

14. The Afonin Lab at UNC Charlotte
Dr. Kirill Afonin
Dr. Caryn Striplin
Dr. Brittany Johnson
Dr. Martin Panigaj
Justin Halman
Weina Ke
Kyle Roark
Sameer Sajja
Oleg Shevchenko
Shriram Jawahar
Strahinja Talic
Allison Tran
Melina Richardson
Rosalie Truong
Jessica McMillan
Dana Elasmar
Acknowledgements
Collaborator Contributions
UNC Charlotte: Marcus Jones, Ian Marriott, Jose Castaneda
NC State University: Yaroslava G. Yingling, Joseph Tracy, Ho Shin Kim
College of William & Mary: Kristin L. Wustholz, Jenna A. Tan
Leidos Biomedical Research / Frederick National Laboratory: Mathias Viard
NIH R01GM120487