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Rebecca E. Thorne
B.Sc., Simon Fraser University, 2002



In the Department of
Molecular Biology and Biochemistry

O Rebecca E. Thorne 2005


Fall 2005

All rights reserved. This work may not be
reproduced in whole or in part, by photocopy

or other means, without permission of the author

Page 2


Name: Rebecca E. Thorne

Degree: Master of Science

Title of Thesis:

Examining Committee:


A Serotonin-Dependent Deoxyribozyme that uses light to
repair thymine dimers in DNA.

Dr. Fiona Brinkrnan
Assistant Professor, Department of Molecular Biology and
Biochemistry, SFU

Dr. Dipankar Sen
Senior Supervisor
Professor, Department of Molecular Biology and
Biochemistry, SFU

Dr. Andrew Bennet
Committee Member
Professor, Department of Chemistry, SFU

Dr. Peter Unrau
Committee Member
Assistant Professor, Department of Molecular Biology and
Biochemistry, SFU

Dr. Melanie O'Neill
Internal Examiner
Assistant Professor, Department of Chemistry, SFU

Date DefendedIApproved: November 14.2005

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another broad peak of absorbance ranging from 290 nm to 3 10 nm. This later peak was of

more interest since we can excite serotonin selectively in the presence of the

deoxyribozyme at greater wavelengths than 300 nm. But, is exciting serotonin at

wavelengths greater than 300 nm better for catalysis? I wanted to determine an optimal

wavelength of catalysis for Serol C, therefore I investigated the wavelength dependence

of SerolC catalysis to produce an action spectrum.

I used a spectrofluorimeter to excite the serotonin, within the reactions, at

different wavelengths. Before creating a final action spectrum (wavelength dependence

of Serol C), I performed experiments to correct for the different values of light intensity

at each wavelength. One of the best chemical actinometers, to do so, is the potassium

ferrioxalate (K3Fe(C204)3) system developed in the early 1950s (Hatchard and Parker

1956). Upon irradiation of (K3Fe(C204)3) solutions the Fen' reduces to Fen. The more

light intensity being directed at the sample, the more Fe"' will reduce to Fe". A 1,lO-

phenanthroline solution is then added, and the formation of [ ~ e ( ~ h e n ) ] " is then detected

via absorption at 510 nm. Therefore, the more [ ~ e ( ~ h e n ) ] " detected, the greater the light

intensity at that particular wavelength. Figure 2-7 shows that the light intensity is

generally increasing as I increased the wavelength of irradiation. These values are

expected with the use of the Xenon Short Arc Lamp used in our spectrofluorimeter. My

values obtained for this experiment on our spectrofluorimeter, shown in Figure 2-7, were

used to determine correction factors to standardize the initial rate values obtained in the

analysis of the reactions at each wavelength, such that we then assumed constant light

intensity for each wavelength.

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Absorbance @ 510nm
of Fe(ll)-phenanthroline

225 250 275 300 325 350 375
Wavelength (nm)

Figure 2-7 Potassium ferrioxalate actinometry analysis; monitoring production of Fe(I1) at 510nm.

The final action spectrum of SerolC with serotonin is shown in Figure 2-8, and

indicates that the optimal wavelength for repair of the thymine dimer substrate by the

deoxyribozyme, and not by direct photoreversal, was at 300 nrn. Again, both negative

controls were performed to ensure that direct photoreversal, or direct repair by serotonin

were not playing a significant role in the catalysis. These confirm that catalysis requires

both by SerolC and serotonin (Figure 2-8). However, the rate enhancements plotted on

top of the action spectrum (Figure 2-9) shows that the optimal wavelength for rate

enhancement, over the background rates, was at approximately 3 15 nrn. Therefore, even

where serotonin naturally absorbs the most amount of light energy, at 285 nm, there was

little rate enhancement over the background reactions at this wavelength.

Page 120

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