A Multi-Method Directed Evolution to Create Improved Far-Red Fluorescent Proteins
Open Access DepositedFluorescent proteins (FPs) have revolutionized the field of molecular and cellular biology by enabling the visualization of dynamic biological processes with unprecedented precision and clarity. The development of new and improved FPs through directed evolution has been a thriving area of research in recent years. This dissertation explores the vast potential of directed evolution techniques in tailoring FPs for diverse and advanced imaging applications.The primary objective of this research is to harness the power of directed evolution to engineer FPs with enhanced characteristics, including brightness, photostability, and spectral diversity. By combining rational design principles with high-throughput screening methodologies, we have developed multiple systematic approaches for the directed evolution of FPs. These methodologies involve the creation of diverse FP libraries, followed by rigorous selection and screening processes, which ultimately lead to the identification of novel FPs with superior properties in varying biological systems. Chapter 1 provides background information on the discovery and evolution of fluorescent proteins over the years and the continued effort to expand the number and colors of fluorescent proteins. The emphasis is on small ultra red fluorescent protein (smURFP), which is a far-red fluorescent protein, and the ways the biophysical characteristics of the protein were improved using directed evolution. Chapter 2 describes the traditional method of evolving fluorescent proteins in bacteria, which was used to evolve small ultra red fluorescent proteins (smURFP). The emphasis is on the methodology and how it can be used to evolve different fluorescent proteins in bacteria to optimize the biophysical characteristics of the protein. Chapter 3 describes a rapid method for evolving fluorescent proteins in mammalian cells using a novel method we developed. The method eliminates multiple time-consuming microbiology steps, which are done in more traditional methods, and it allows the fluorescent proteins to be evolved in mammalian cells directly. Here, the new method was used to evolve smURP, and a new class of fluorescent proteins, Serpin, to be brighter in mammalian cells without the addition of a fluorophore. Chapter 4 describes a directed evolution method for evolving smURFP in mammalian cells utilizing error-prone polymerase chain reaction (PCR) and fluorescence activated cell sorting (FACS). This methods allows for smURFP to be evolved directly in mammalian cells for optimization. smURFP went through four iterative rounds of directed evolution to create a variant that is 12-fold brighter than smURFP WT. The new variants biophysical properties were tested against smURFP WT, and were compared by fluorescent imaging against other NIR fluorescent proteins by fluorescent imaging. Chapter 5 investigates a novel approach that integrates Matrix-Assisted Laser Desorption Ionization (MALDI) and Silicon Nanopost Array Laser Desorption Ionization (NAPA-LDI) for quantitative imaging and enhanced molecular coverage. Our study involves the placement of tissue sections on a NAPA substrate, followed by the deposition of matrix and/or standards, resulting in superior molecular coverage achieved through low-fluence MALDI and high-fluence NAPA-LDI.
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