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Specificity and stoichiometry of the G-quadruplex binding protein BG4

Johnson, Samuel Arthur (2023) Specificity and stoichiometry of the G-quadruplex binding protein BG4. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

A G-quadruplex (G4) is a non-canonical secondary structure that may form in single-stranded nucleic acids with repetitive guanine-rich sequences, or putative quadruplex sequences (PQSs). Consisting of stacked G-quartets held together by Hoogsteen bonding and stabilized by central monovalent cations, G-quadruplexes are highly thermostable and can prevent annealing or other base-specific binding by proteins and nucleic acids. PQSs exist throughout the mammalian genome, notably in the repetitive TTAGGG telomeric sequence and in gene promoters. G-quadruplexes are therefore proposed to have diverse biological functions, including pre- and post-transcriptional gene regulation and telomere protection, but studying them in cellular contexts is difficult due to limited detection options and the variety of conformations that a PQS may assume. The leading method for identifying G-quadruplexes in cells is BG4, a 32.7-kDa single-chain variable fragment (scFv) antibody developed to bind to many quadruplex topologies with high specificity over non-quadruplex structures. Here, we further characterize BG4’s binding specificity and stoichiometry using a combination of biochemical and biophysical methods. We observe that BG4 is capable of binding to PQS oligonucleotides containing alterations that prevent stable G-quadruplex formation, showing that BG4 binding is more flexible than originally hypothesized. We also present evidence that BG4 may promote and stabilize G-quadruplex folding, a characteristic of other G4-binding reagents that had not been observed for BG4, and demonstrate that BG4 binds its G-quadruplex targets with 1:1 stoichiometry. These are critical insights for understanding BG4 may be effectively used to study the formation and functions of G-quadruplexes in cells.

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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Johnson, Samuel Arthursaj98@pitt.edusaj980000-0002-0015-8866
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Thesis AdvisorOpresko, Patriciaplo4@pitt.eduplo40000-0002-6470-2189
Committee ChairVan Houten, Bennettvanhoutenb@upmc.edu0000-0002-4009-2478
Committee MemberAlder, Jonathanjalder@pitt.edujalder0000-0003-3741-6512
Committee MemberMyong, Suasmyong1@jhu.edu
Committee MemberZhang, Huiayinghuaiyinz@andrew.cmu.edu0000-0002-1784-2664
Date: 29 September 2023
Date Type: Publication
Defense Date: 16 March 2023
Approval Date: 29 September 2023
Submission Date: 24 April 2023
Access Restriction: 1 year -- Restrict access to University of Pittsburgh for a period of 1 year.
Number of Pages: 138
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Molecular Biophysics and Structural Biology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: G-quadruplex, BG4, telomere
Date Deposited: 29 Sep 2023 16:12
Last Modified: 29 Sep 2023 16:12
URI: http://d-scholarship.pitt.edu/id/eprint/44703

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