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Optical and small-molecule control of protein and nucleic acid function

Wesalo, Joshua Scott (2023) Optical and small-molecule control of protein and nucleic acid function. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Nature carries out its molecular biology work with the utmost of spatial and temporal control. To study the underlying processes, investigators need tools that can switch biomolecules on or off at will, quickly, and decisively. Biological settings are compartmentalized and complex, and researchers must develop methods that can reach specific targets and function in their surroundings without interference. Herein, I describe my contributions toward several tools that meet these challenges. Most of these tools are based on unnatural amino acids (UAAs), and the accompanying translational machinery to install them site-specifically into proteins. I first describe optimization of the machinery, then present several new UAAs. These include masked residues that respond to bioorthogonal small-molecule triggers or light. The UAAs presented here unmask with unprecedented speed in live cells. Many of them are optimized azido-UAAs that respond to phosphine triggers via the Staudinger reduction. This chemistry is exceptionally bioorthogonal, but was previously limited by slow kinetics. I describe azide-masked tyrosine and lysine residues with accelerated activation rates. Additionally, I report several new UAAs that respond to light, including lysine and histidine analogues. Many of these UAAs can work together in demanding, multiplexed experiments, either sequentially or orthogonally. I have leveraged these tools to control protein subcellular localization, to study SUMOylation, and to control enzyme function (including bioluminescent enzymes and a recombinase).
In addition to these UAAs, I have also developed an approach for using a modified Staudinger ligation to trigger gene knockdown with an azide trigger. This is performed using a circularized morpholino oligonucleotide that blocks translation once an azide cleaves it to linearize it. These tools expand the experimental options available for probing protein and nucleic acid biology in live cells and organisms.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Wesalo, Joshua Scottjosh.wesalo@gmail.comJSW510000-0003-4389-8976
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairDeiters,
Committee MemberWeber, Stephen
Committee MemberChilders, W.
Committee MemberGross, Jeffrey
Date: 11 May 2023
Date Type: Publication
Defense Date: 21 July 2021
Approval Date: 11 May 2023
Submission Date: 4 April 2023
Access Restriction: 2 year -- Restrict access to University of Pittsburgh for a period of 2 years.
Number of Pages: 441
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Chemistry
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: Bioorthogonal chemistry, Staudinger ligation, unnatural amino acids, photocaged, genetic code expansion, chemical biology
Date Deposited: 11 May 2023 19:16
Last Modified: 11 May 2023 19:16


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