STUDIES IN FLUORESCENCE RESONONANCE ENERGY TRANSFER IN
A FAM/IOWA BLACK LABELED THROMBIN-BINDING APTAMER
Jay Blatnik
UW-Whitewater
Jay Blatnik and Paul G. House
University of Wisconsin–Whitewater, Department of Chemistry
Whitewater, Wisconsin
Background
Study Design
TBA folds into a unique “chair” structure when
it binds to thrombin or to certain cations. TBA’s
interaction with the cations may allow for a better
understanding of the its folding characteristics. In
the chair structure TBA has two guaninequadruplexes that are arrangements of 4 guanines
stabilized by hydrogen bonds. G-quadruplexes
have received much attention because guanine rich
sequences are commonly found in the human
genome and have mechanisms linked to cancer,
HIV and other diseases.
Our previous work with TBA dual labeled with
FAM and TAMRA on the 3’ and 5’ ends, is graphed
below. It shows an increase in fluorescence with the
addition of monovalent cations but a decrease in
fluorescence with the addition of divalent cations.
These results are inconsistent with work from other
researchers who have shown both K+ and Sr2+ cause
TBA to fold. These inconsistent results led us to use
a differently labeled aptamer. Iowa black is an
acceptor that leads to decreased fluorescence when
FRET occurs.
Fluorescence resonance energy transfer (FRET)
is the transfer of energy from a donor to an acceptor
and is distance dependant.
F-TBA-T Time Dependant
100
80
60
KCl
40
NaCl Initial Trial
20
NaCl Second Trial
0
SrCl2
-20 0
50
100
150
BaCl2
-40
NH4Cl
-60
-80
Volume of added salt solution (μL)
Results for FAM/TAMRA labeled probe with a 10 minute wait
between scans (λEX: 495.0 nm, monitored at 577.0 nm and 5.0
nm slit widths)
FAM/Iowa black TBA
Corrected Fluorescence
We monitored the folding of TBA with a variety
of cations including previously well studied (Na+, K+)
and two seldom looked at (Rb+, Cs+). All
experiments were conducted at room temperature.
Data was corrected for the increase in volume as the
salt solution was added; the percent difference in
fluorescence was calculated and graphed.
% Difference
Aptamers are single strands of DNA or RNA 15
to 50 bases long that bind strongly and specifically
to a molecular target. These molecules are
increasingly popular in diagnostics, research and
therapeutics. Thrombin-binding aptamer (TBA) is
one aptamer getting a lot of attention. Thrombin is
a protein important in blood coagulation.
Mentor: Dr. Paul House
UW-Whitewater
250.0
200.0
150.0
LiCl
CsCl
KCl
NaCl
LiCl 2
100.0
50.0
0.0
0.00
1.00
2.00
3.00
[Salt] (mM)
Results of FAM/Iowa Black-labeled probe with a 10
minute wait between scans (λEX: 495.0 nm, monitored
from 500.0 to 600.0 nm and 5.0 nm slit widths)
Discussion & Future
Experiments.
Using a dual labeled aptamer containing the
quencher Iowa black we expected a decrease in
fluorescence as the aptamer folded. Instead we saw
an increase in fluorescence. In addition, the
absolute intensity of the fluorescence fluctuated
between experiments.
A number of possible experimental factors
including instrumental settings (excitation and
detection wavelength and monochromator slit
width) were examined to explain these results ,
nothing could explain for the increase in
fluorescence as the distance decreased between the
donor and quencher
In future studies we plan to use a modified
TBA (CCaaCggTTggTgTggTTgg) dual labeled with
FAM and Dabcyl. The delay between scans will be
performed submerged in heated water to ensure for
complete unfolding.
Summary
Hypotheses
Using a dual labeled TBA, as ions are added to
the solution total fluorescence will decrease when
using FAM and Iowa Black-FQ as a result of FRET.
A dual labeled thrombin binding aptamer
produced an increase in fluorescence for ions that
other researchers have shown should bring the
donor/acceptor pair FAM/Iowa black closer
together. This is not expected – Iowa black should
decrease the FAM fluorescence. Careful study of
how the experiment was done did not reveal the
cause of this inconsistency.
Acknowledgements:
•UW-Whitewater Undergraduate
Research Program
•Dr. Paul House
•Ms. Marsha Goodell