Discovery of novel functional DNA and their application as biological sensors

Lake, Ryan James

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https://hdl.handle.net/2142/115659 Copy

Description

Title

Discovery of novel functional DNA and their application as biological sensors

Author(s)

Lake, Ryan James

Issue Date

2022-03-18

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

Lu, Yi

Doctoral Committee Chair(s)

Lu, Yi

Committee Member(s)

• Cheng, Jianjun
• Hergenrother, Paul J
• Zimmerman, Steven C

Department of Study

Chemistry

Discipline

Chemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• functional DNA
• DNAzymes
• aptamers
• iron sensor
• SARS-CoV-2
• deoxyriboswitch

Abstract

Functional DNA are DNA molecules that are able to perform some sort of function by folding into a specific conformation based on their nucleotide sequence, analogous to ribozymes and riboswitches. They are identified from a combinatorial chemistry technique called in vitro selection in which a randomly synthesized pool of DNA is selected over multiple rounds to carry out the desired function. These functional DNA can perform various functions, such as binding to a certain target, called a DNA aptamer, or catalyzing a specific chemical reaction, called a DNAzyme. Because of the high stability and biocompatibility of DNA, many of these functional DNA have been developed toward various applications in biological systems, especially for use as sensors. For my thesis project, I undertook three separate projects that all involved application or discovery of functional DNA in relationship to biological systems. In the first two projects, I developed novel functional DNA into sensors that could be used for detection of a certain analyte within the context of biology. In the third project, I attempted to identify functional DNA that is native to biological systems. Metal ions play important roles in various aspects of biology, and iron plays an especially important role as the most abundant transition metal in biology. Furthermore, misregulation of iron has been implicated in a wide variety of diseases, including various cancers and neurological diseases. As such, it has become clear that methods of detecting iron in biological systems, especially within labile pools, could be important to better understand the role of iron in these processes. In this project, I developed two different RNA-cleaving DNAzymes into fluorescent turn-on sensors, based on Fe3+- and an Fe2+-dependent DNAzymes, using the catalytic beacon approach. These sensors can detect their analyte with high specificity over the other oxidation state, down to the micromolar level. I then demonstrated that these sensors could be used to detect changes in intracellular Fe3+ and Fe2+ labile pools simultaneously, due to incubation with transferrin-bound iron, as well as in a model of ferroptosis, which had previously been shown to be involved with misregulation of iron homeostasis. Viral infections are a major global health issue, but no current method allows rapid, direct, and ultrasensitive quantification of intact viruses with the ability to distinguish infectious from non-infectious virus, allowing for the continued spread of viral diseases. In this project, I developed a method for direct detection and differentiation of infectious from noninfectious SARS-CoV-2, as well as differentiation from other coronaviruses, without any sample pretreatment. DNA aptamers were selected from a DNA library to bind to intact, infectious virus but not to inactivated virus, which were then incorporated into a solid state nanopore, which allows strong confinement of the virus to enhance sensitivity down to 1×104 copies/mL of SARS-CoV-2. Our method matches the sensitivity of plaque assays for direct viral detection, while also differentiating infectious from non-infectious virus and taking a much shorter time to obtain results. Though a wide variety of functional DNA molecules have been identified in the lab from synthesized DNA pools, there has yet to be discovered any functional DNA sequences that natively play a role in biology. I hypothesized that one potential role that such a functional DNA sequence could play would be as a “deoxyriboswitch,” analogous to riboswitches, that would be able to regulate expression of certain genes in the presence of a certain analyte. In this project, I used Genomic Selection and bioinformatic searching of existing aptamers to identify two sequences that can bind to ATP in vitro: one from bacteria and another from humans. I further obtained some preliminary results that suggest that the bacterial sequence may be able to regulate transcription levels in vitro. However, more studies are needed to definitively determine if these sequences are able to function as gene regulators in cells.

Graduation Semester

2022-05

Type of Resource

Thesis

Copyright and License Information

Copyright 2022 Ryan Lake

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