Short-Range Single-Molecule FRET

Using a Contact Quenching Approach

Callum Johnston Benjamin Ambrose Timothy Craggs

AIMS  Establish a repeatable and reliable method for quantitative quenchable FRET (qqFRET).
 Study the impact of macro and micro-solvents on qqFRET.
THEORY
2) smFRET
1) FRET
 Förster Resonance Energy Transfer  Instead of ensemble averaging, smFRET detects one
(FRET) is a radiationless transmission of molecule at a time – providing the real distribution
energy from a donor to an acceptor (right, top). [2]
molecule. [1]  Samples are labelled with donor (D) and acceptor
 FRET operates in the range of 3 – 10 (A) fluorophores.
nm.  These are exited at specific wavelengths using
 The FRET efficiency is linked to the Alternating Laser Excitation (ALEX) which allows
distance between fluorophores (below) exclusion of inaccurate measurements.
 Carried out using confocal microscopy on the
𝟏 𝑫𝑨 Cragg’s lab laser (right, bottom):
𝑬= 𝑬 ∗=
𝑫𝑫 + 𝑫𝑨
(𝟏)+ ¿ ¿  Limitations of smFRET: molecules can conform on a
scale lower than 3 nm (e.g. HIV-1 protease).

3) qqFRET
 qqFRET is a novel approach to short-range
smFRET.
 Distances between 1 – 3 nm can in theory be
measured.
 This method quantifies the degree of quenching
(magnitude of Q value) instead of whether
quenching is simply occurring.
 Relatively new technique used by Cragg’s lab to
study structure specific DNA binding.
Quenched
4) AV clouds
 The accepter is placed close enough to quench
both donor fluorescence and FRET – the donor
can no longer be excited and therefore cannot
FRET to acceptor. [3]
 They’re positioned at low to mid to high
overlap (top left).
 The AV clouds plot all the points the dyes can
physically reach once attached, as a series of 3D
coordinates (bottom left).
Unquenched  At these short ranges, the dyes may undergo
contact quenching.

RESULTS
3) Q relation to viscosity
1) Repeatability of prior data set  A theoretical model (bottom left, work by Ben Ambrose)
predicts a lower Q value with a increase in viscosity of the
 A data set recorded prior to this project was buffer.
repeated to give a similar trend line (right).
 A viscous solution should reduce the chance of the donor
 Results show that the amount of quenching varies fluorophore quenching the acceptor.
with the AV cloud overlap.
 Initial results show a significant difference between a 50%
glycerol buffer solution (blue) compared to one with no
glycerol (red).
2) Q relation to laser power
 The Q value you measure is intrinsically linked to No Glycerol
the red laser power.

 An increase in the laser power gives a Q value that

more closely resembles the original data set. 50% Glycerol

 A higher red laser power picks up the quenched dye

photon emission more efficiently than before, since
the quenched dye emits light of a lower
intensity.

CONCLUSIONS FUTURE WORK

 The method for qqFRET is repeatable and garners consistent results that correlate  Change the percentage of glycerol in the
with expected results. buffer to investigate the effect of different
degrees of viscosity.
 The link between the green and red laser powers and the quenching coefficient was
studied and the method subsequently changed / improved to account for any error.  Apply the qqFRET method to study the
kinetics and conformations of DNA hairpins
 The degree of quenching between the fluorophores can be altered by changing the (right).
viscosity of the buffer solution.

ACKNOLEDGEMENTS REFERENCES
[1] T. Förster, Ann. Phys., 1948, 437, pp. 55–75.
I’d like to thank Dr. Craggs for his supervision
[2] T. Cordes, Y. Santoso, A. I. Tomescu, K. Gryte, L. C. Hwang, B. Camará, S. Wigneshweraraj and A. N. Kapanidis,
as well as Ben Ambrose and the rest of the Biochemistry, 2010, 49, pp. 9171–9180.
Craggs/Cadby group. [3] Ingargiola, A., Lerner, E., Chung, S. Y., Weiss, S., & Michalet, X. PLoS One, 2016, 11 (8), pp. 1-25.