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Visualizing dynamics of the central dogma in living single cells

Guo, Yue (2022) Visualizing dynamics of the central dogma in living single cells. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Imaging the central dogma at single molecule resolution in single cells reveals the spatial and temporal dynamics and ubiquitous cell-to-cell variability of molecular events. In my dissertation work, I first implement SunTag-based translation reporter and quantify the copy numbers of signaling molecule NEMO in NF-κB pathway to illustrate the use of state-of-the-art single molecule approach to study the central dogma. Transcription process to produce mRNAs as the start of the central dogma is the chief regulator of gene expression. However, most approaches to image mRNAs require cell fixation or have limited single-molecule sensitivity for live-cell applications. I therefore develop and characterize SunRISER, an approach for long-term imaging of mRNA in living cells. SunRISER employs SunTag as a scaffold to achieve fluorescence signal amplification of coat proteins and enhance contrast of mRNAs. Although the naïve design is impractical, with inconsistent fluorescent properties that complicate mRNA detection, I optimize the approach using computational and synthetic biology to achieve robust and unambiguous detection of individual mRNAs. SunRISER-labeled mRNAs are resistant to photobleaching and the design principle is generalizable for robust whole-cell mRNA imaging experiments with orthogonal tagging systems. SunRISER variants using shorter 8x and 10x stem-loop arrays (SunRISER SRv1.1 and SRv1.2, respectively) also result in consistent mRNA labeling and detection, while reducing the size of alterations to target mRNA sequences. As an application of SunRISER, I interrogate mitotic inheritance of mRNA molecules during a variety of stresses. When observed over the period of cell doubling time, it is found that mitotic mRNA inheritance is equally partitioned in standard growth conditions and that inflammatory stress or nutrient limitation can enhance diversity among post-mitotic sister cells. SunRISER can be applied to other RNA species with further modification. Taken together, SunRISER enables a window into living cells to observe aspects of the central dogma in addition to roles of mRNAs in rare and dynamical trafficking events.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Guo, Yueyug31@pitt.eduyug31
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairWu,
Committee CoChairLee, Robin E.
Committee MemberSwanson,
Committee MemberXu,
Committee MemberSalman,
Date: 11 October 2022
Date Type: Publication
Defense Date: 21 July 2022
Approval Date: 11 October 2022
Submission Date: 5 August 2022
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Number of Pages: 145
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Physics
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: single mRNA; live cell imaging; SunTag
Date Deposited: 11 Oct 2022 19:49
Last Modified: 11 Oct 2022 19:49

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