Alternative signal transduction for functional nucleic acid sensors

Pawel, Gregory T.

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https://hdl.handle.net/2142/117541 Copy

Description

Title

Alternative signal transduction for functional nucleic acid sensors

Author(s)

Pawel, Gregory T.

Issue Date

2022-12-01

Director of Research (if dissertation) or Advisor (if thesis)

Lu, Yi

Doctoral Committee Chair(s)

Lu, Yi

Committee Member(s)

• Murphy, Cathy
• Zhao, Huimin
• Zhang, Kai

Department of Study

Chemistry

Discipline

Chemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• DNA
• Sensors
• Microscopy
• Fluorescence
• Signal Transduction

Abstract

A good sensor has two requirements: 1) analyte recognition and 2) signal transduction. This means that a sensor must correctly identify a molecule distinct from the environment and then report that in a measurable way. DNA aptamers, which are artificially created DNA polymers that have been engineered to have binding affinity for a specific target, are particularly suited for both. To be successful in analyte recognition, the sensor must be sensitive, selective, and safe. Through the process of in vitro selection, a DNA aptamer can be made to have very strong binding affinity to a target molecule, i.e., it is sensitive. Similarly, by using counterselections, aptamers can be made to have no activity towards similar molecules, i.e., it is selective. Last, DNA is naturally biocompatible and non-toxic, and aptamer-target interactions are generally reversible, which makes them safe in most any circumstance. Because of advancements in solid phase synthesis of DNA, there are many modifications into DNA which can be incorporated. This means that DNA aptamers can be outfitted with any number of other functional groups which can act as signal transduction for a sensor. The most popular type of modification is to add a fluorescent molecule onto the DNA. When paired with a sensor, this can create a fluorescent turn-on signal when the aptamer binds to a target. In this way, the aptamer acts as both the analyte recognition as well as a scaffold for the signal transduction. There are also many other types of modifications that can be used to conjugate the DNA to other molecules like nanoparticles, proteins, or bulk surfaces. In this dissertation I present several research projects spanning the creation of new DNA aptamers (ch.3), characterization of the binding ability of aptamers (ch.3, ch.4, ch.5), engineering of aptamers into new types of sensors (ch.3, ch.6, ch.7), and the implementation of these sensors in new ways for cellular imaging (ch.2, ch.7). Specifically, chapter 2 presents an already published paper wherein we use an ATP-binding aptamer to detect cellular ATP specifically at the mitochondria in living cells. We use nanoparticles called DQAsomes as a delivery method to deliver our fluorescent sensors directly to the mitochondria, where the activity is measured using microscopy. Chapter 3 presents another already published work. In this Science Advances paper, we select two new aptamers- one for the human adenovirus and one for the novel coronavirus SARS-CoV-2 which ravaged the global economy in 2020. After selecting the aptamer, we inserted it into a nanopore in order to electrochemically detect extremely small concentration of virus. In addition, these aptamers show the unique ability to distinguish between active virus and non-infectious forms of the same virus, which is something that PCR cannot do. Chapter 4 is a soon-to-be published bioprotocol paper which was requested of use by readers of chapter 3. It contains a step-by-step instruction of how to perform 2 types of aptamer characterization experiments specifically for viruses, which pose different biosafety challenges from other small molecule targets. Chapter 5 catalogs a long list of aptamer characterization using isothermal titration calorimetry in our quest to find naturally occurring DNA aptamers. After testing many different DNA candidates and a number of target possibilities we found no definitive aptamers, but a few candidates have moved on to further testing. Chapter 6 attempts to shed light on an old problem. Metal ions in solution interfere with fluorescent sensors because they can quench fluorescence. In order to solve that problem, I studied the mechanism of how those metal ions quench fluorescence. It is determined that the mechanism of quenching is largely dependent on both the DNA being used as well as the choice of fluorophore. Chapter 7 is an attempt to act on the information from chapter 6 to solve the quenching problem. Because fluorescence intensity-based sensors are vulnerable to environmental effects, I engineered a sensor to have different modes of signal transduction- namely fluorescence lifetime and fluorescence anisotropy. Importantly, I applied this sensor in cells and used all 3 detection modes simultaneously, something that has not been done for DNA sensors before, in order to discover the strengths and weaknesses of each method.

Graduation Semester

2022-12

Type of Resource

Thesis

Copyright and License Information

Copyright 2022 Greg Pawel

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