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Engineering Nucleic Acid Mechanisms for Regulation and Readout of Gene Expression: Conditional Dicer Substrate Formation and Sensitive Multiplexed Northern Blots

Citation

Schwarzkopf, Ma'ayan (2013) Engineering Nucleic Acid Mechanisms for Regulation and Readout of Gene Expression: Conditional Dicer Substrate Formation and Sensitive Multiplexed Northern Blots. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/Z94Q7S0V. https://resolver.caltech.edu/CaltechTHESIS:05212013-161536447

Abstract

Nucleic acids are most commonly associated with the genetic code, transcription and gene expression. Recently, interest has grown in engineering nucleic acids for biological applications such as controlling or detecting gene expression. The natural presence and functionality of nucleic acids within living organisms coupled with their thermodynamic properties of base-pairing make them ideal for interfacing (and possibly altering) biological systems. We use engineered small conditional RNA or DNA (scRNA, scDNA, respectively) molecules to control and detect gene expression. Three novel systems are presented: two for conditional down-regulation of gene expression via RNA interference (RNAi) and a third system for simultaneous sensitive detection of multiple RNAs using labeled scRNAs.

RNAi is a powerful tool to study genetic circuits by knocking down a gene of interest. RNAi executes the logic: If gene Y is detected, silence gene Y. The fact that detection and silencing are restricted to the same gene means that RNAi is constitutively on. This poses a significant limitation when spatiotemporal control is needed. In this work, we engineered small nucleic acid molecules that execute the logic: If mRNA X is detected, form a Dicer substrate that targets independent mRNA Y for silencing. This is a step towards implementing the logic of conditional RNAi: If gene X is detected, silence gene Y. We use scRNAs and scDNAs to engineer signal transduction cascades that produce an RNAi effector molecule in response to hybridization to a nucleic acid target X. The first mechanism is solely based on hybridization cascades and uses scRNAs to produce a double-stranded RNA (dsRNA) Dicer substrate against target gene Y. The second mechanism is based on hybridization of scDNAs to detect a nucleic acid target and produce a template for transcription of a short hairpin RNA (shRNA) Dicer substrate against target gene Y. Test-tube studies for both mechanisms demonstrate that the output Dicer substrate is produced predominantly in the presence of a correct input target and is cleaved by Dicer to produce a small interfering RNA (siRNA). Both output products can lead to gene knockdown in tissue culture. To date, signal transduction is not observed in cells; possible reasons are explored.

Signal transduction cascades are composed of multiple scRNAs (or scDNAs). The need to study multiple molecules simultaneously has motivated the development of a highly sensitive method for multiplexed northern blots. The core technology of our system is the utilization of a hybridization chain reaction (HCR) of scRNAs as the detection signal for a northern blot. To achieve multiplexing (simultaneous detection of multiple genes), we use fluorescently tagged scRNAs. Moreover, by using radioactive labeling of scRNAs, the system exhibits a five-fold increase, compared to the literature, in detection sensitivity. Sensitive multiplexed northern blot detection provides an avenue for exploring the fate of scRNAs and scDNAs in tissue culture.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:nucleic acid engineering; RNAi; RNA interference; northern blot; hybridization chain reaction; HCR; nanotechnology; conditional RNAi; scRNA; scDNA
Degree Grantor:California Institute of Technology
Division:Biology
Major Option:Biology
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Pierce, Niles A.
Thesis Committee:
  • Deshaies, Raymond Joseph (chair)
  • Baltimore, David L.
  • Hay, Bruce A.
  • Pierce, Niles A.
Defense Date:9 May 2013
Record Number:CaltechTHESIS:05212013-161536447
Persistent URL:https://resolver.caltech.edu/CaltechTHESIS:05212013-161536447
DOI:10.7907/Z94Q7S0V
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:7733
Collection:CaltechTHESIS
Deposited By: Maayan Schwarzkopf
Deposited On:19 Jan 2016 22:08
Last Modified:08 Nov 2023 00:43

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