Development of a Variable Soluble Protein Detector with Cas12a Amplification Open Access

Abstract

The field of proteomics is in need of a rapid, simple, and sensitive protein detection method, with current methods being both costly and time intensive. Cas12a is an RNA guided endonuclease found within the CRISPR-Cas family of enzymes present in bacteria and archaea that has the ability to both cleave and degrade foreign nucleic acids. This cleavage ability has previously been shown to have the potential to improve biosensing and detection assays, but many of Cas12a’s mechanisms within these biosensing assays remain poorly characterized. This thesis focuses on the development of a soluble protein detector that utilizes Cas12a amplification. The detector (VASA) is a 4 oligomer complex with a double stranded region that conceals a Cas12a activator strand and 2 single stranded regions that can fold into aptamers relevant to the test protein, human α-thrombin. In order to activate Cas12a, the binding of VASA to α-thrombin needs to generate a force that ruptures VASA, revealing the Cas12a activator strand. Experiments were conducted to characterize VASA, confirm binding interactions, and understand the parameters by which VASA can mechanically rupture and activate Cas12a amplification. Four distinct protein-attractive platforms were also tested for their ability to mechanically rupture VASA. Our investigations have led to a greater understanding of Cas12a behavior and requirements for VASA rupturing.

Table of Contents

Chapter 1. Introduction

1.1 A History of Protein Detection and Identification ---------------------------------------------- 2

1.2 STRETCH and Its Implications for Protein Detection ------------------------------------------ 4

1.3 Selection of a Soluble Protein Target: Human α-Thrombin ----------------------------------- 7

1.4 Overview of Goals ----------------------------------------------------------------------------- 8

Chapter 2.Design and Characterization of an Oligomer Detector

2.1 Background and Design -------------------------------------------------------------------------- 11

2.2 Methods ----------------------------------------------------------------------------------------- 12

2.3 Results ------------------------------------------------------------------------------------------- 17

2.4 Discussion --------------------------------------------------------------------------------------- 23

2.5 Conclusion -------------------------------------------------------------------------------------- 25

Chapter 3. Confirmation of Simultaneous Binding of Aptamers and α-Thrombin

3.1 Background and Design -------------------------------------------------------------------------- 27

3.2 Methods ----------------------------------------------------------------------------------------- 29

3.3 Results ------------------------------------------------------------------------------------------- 31

3.4 Discussions-------------------------------------------------------------------------------------- 33

3.4 Conclusion -------------------------------------------------------------------------------------- 33

Chapter 4. The Search for a VASA Rupturing Opener

4.1 Background and Overall Design ----------------------------------------------------------------- 35

4.2 Simple Opener Cas12a Mediated VASA Rupturing ---------------------------------------------- 35

4.3 AuNP Cas12a Mediated VASA Rupturing -------------------------------------------------------- 40

4.4 Worm-like Chain Mediated VASA Rupturing ---------------------------------------------------- 47

4.5 DNA Origami Mediated VASA Rupturing ------------------------------------------------------- 53

4.6 Conclusion--------------------------------------------------------------------------------------- 60

Chapter 5. Conclusion------------------------------------------------------------------------------- 62

Supplemental Information -------------------------------------------------------------------------- 64

References ------------------------------------------------------------------------------------------- 69

About this Honors Thesis

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School	Emory College
Department	Chemistry
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Chemistry, Biochemistry
Keyword	Cas12a
Committee Chair / Thesis Advisor	Khalid Salaita, Emory University
Committee Members	Simbarashe Nkomo, Emory University David Lynn, Emory University

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