Next-generation oligonucleotide probes for transcriptome in vivo analysis (TIVA) / Sean B. Yeldell.

Format:

Book

Manuscript

Thesis/Dissertation

Author/Creator:

Yeldell, Sean B., author.

Contributor:

Dmochowski, Ivan J., degree supervisor.

Chenoweth, David M., degree committee member.

Cooperman, Barry S., degree committee member.

Tsourkas, Andrew, degree committee member.

University of Pennsylvania. Department of Chemistry, degree granting institution.

Language:

English

Subjects (All):

Penn dissertations--Chemistry.

Chemistry--Penn dissertations.

Local Subjects:

Penn dissertations--Chemistry.

Chemistry--Penn dissertations.

Physical Description:

x, 158 leaves : illustrations ; 29 cm

Production:

[Philadelphia, Pennsylvania] : University of Pennsylvania, 2018.

Summary:

The transcriptome--the total collection of every RNA transcript in a cell--provides a unique readout of a cell's commands as it executes its own genetic code. Studying the transcriptomes of individual cells is a powerful way to identify new cell types, building a better understanding of complex tissues from the ground up. Furthermore, transcriptomic characterization of tumor samples is already leading to tangible advancements in personalized cancer care. However, the process of capturing a transcriptome requires careful isolation of mRNA from a single cell that is potentially entrenched in an entangled, three-dimensional tissue structure.Our lab has previously developed a method to successfully isolate mRNA from single cells still contextualized within living tissue. This method, Transcriptome In Vivo Analysis (TIVA), utilizes a light-activatable oligonucleotide probe to offer fine spatio-temporal control of mRNA capture. TIVA probes are highly modified RNA hairpins or loops incorporating a poly(U) "capture" sequence complementary to the poly(A) tail of mRNA, as well as a biotin moiety to enable pull-down of bound mRNA. To enable spatio-temporal control of mRNA binding, the probes are locked into an inactive "caged" conformation. Laser excitation of the target cell severs photoactivable o-nitrobenzyl- or Ru(II) polypyridyl-based linkages built into the probe, freeing the poly(U) capture sequence to effectively biotinylate mRNA. After photolysis, a small tissue region containing the target cell is aspirated out and lysed so that the target mRNA can be isolated by streptavidin-biotin affinity purification.In this dissertation I present the synthesis, characterization, and application of next-generation TIVA constructs that aim to address various limitations of the original probe. (I) I demonstrate that phosphorothioation extends the serum stability of the TIVA probe from a few minutes to over 24 hours, and mediates uptake into cells without the need for a cell-penetrating peptide. (II) I also present versions of the probe with hairpin-terminating GC pairs, longer blocking strands, and a pegylated hairpin turn which dramatically reduce the probe's pre-photolysis background binding of mRNA. (III) Finally, I show that incorporation of a Ru(II) polypyridyl-based photocleavable linker extends the probe's activation response from 1-photon near-UV light to 2-photon near-IR light. Together, these advancements move TIVA towards a broader range of applications--including deeper regions of challenging, nuclease-abundant tissues--with greater confidence that our construct will remain stable and generate a low-background transcriptome.

Notes:

Ph. D. University of Pennsylvania 2018.

Department: Chemistry.

Supervisor: Ivan J. Dmochowski.

Includes bibliographical references.

OCLC:

1258924131