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DESIGN, CONSTRUCTION, AND CHARACTERIZATION OF THE YSGR MINIMAL CODON FAB LIBRARY FOR CHAPERONE-ASSISTED RNA CRYSTALLOGRAPHY
Title
DESIGN, CONSTRUCTION, AND CHARACTERIZATION OF THE YSGR MINIMAL CODON FAB LIBRARY FOR CHAPERONE-ASSISTED RNA CRYSTALLOGRAPHY.
Creator
Holmes, Sean, Ye, Jingdong, University of Central Florida
Abstract / Description
Of the entire human genome, 90% of all genetic information is transcribed but only a fraction of that subsequent RNA is translated into proteins. RNAs which are not translated into proteins are deemed non-coding RNAs. Little is known about this large category of noncoding RNAs, although they perform a variety of functions within the cell. RNA crystallography is used to study RNA tertiary structure, which gives insight to the function of these non-coding RNAs. However, complications associated...
Show moreOf the entire human genome, 90% of all genetic information is transcribed but only a fraction of that subsequent RNA is translated into proteins. RNAs which are not translated into proteins are deemed non-coding RNAs. Little is known about this large category of noncoding RNAs, although they perform a variety of functions within the cell. RNA crystallography is used to study RNA tertiary structure, which gives insight to the function of these non-coding RNAs. However, complications associated with RNA crystallography arise due to RNA's lack of surface functional group diversity, flexible tertiary structure, and conformational heterogeneity. A novel technique, Chaperone-assisted RNA crystallography (CARC), can greatly improve the success in crystallization of RNA. With this technique, synthetic antibodies called Antigen Binding Fragments (Fabs) are employed as crystallization chaperones to promote the structure elucidation of certain target RNAs. To identify Fabs that complex with RNA molecules of interest, we constructed a randomized library of synthetic antibodies enriched in ligand binding regions with tyrosine, serine, glycine, and arginine residues. This Fab protein library was constructed with a minimal codon design, and then screened against a variety of RNA targets. Several Fabs have been identified and isolated through phage display selection against three RNA targets. These Fabs were expressed and biochemically characterized for their binding affinities and specificities. To date, five Fabs have been identified and they have outstanding capabilities in binding their specific RNA antigen.
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Date Issued
2012
Identifier
CFH0004167, ucf:44856
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFH0004167
VALIDATING DRUG TARGETS THROUGH INHIBITION OF PROTEIN-PROTEIN INTERACTIONS IN MYCOBACTERIUM TUBERCULOSIS
Title
VALIDATING DRUG TARGETS THROUGH INHIBITION OF PROTEIN-PROTEIN INTERACTIONS IN MYCOBACTERIUM TUBERCULOSIS.
Creator
Driscoll, Erin C, Rohde, Kyle, University of Central Florida
Abstract / Description
Tuberculosis is the leading cause of death by single infectious disease worldwide; novel antibiotics are needed to continue to treat this disease. To goal of this project is to provide proof-of-principle support for the idea that targeting protein-protein interactions (PPI) is an appropriate course for the discovery of new drugs. This study optimized the M-PFC assay, which allows detection of PPI in Mycobacteria, through the use of stronger promoters and inducible expression of a peptide...
Show moreTuberculosis is the leading cause of death by single infectious disease worldwide; novel antibiotics are needed to continue to treat this disease. To goal of this project is to provide proof-of-principle support for the idea that targeting protein-protein interactions (PPI) is an appropriate course for the discovery of new drugs. This study optimized the M-PFC assay, which allows detection of PPI in Mycobacteria, through the use of stronger promoters and inducible expression of a peptide blocker by riboswitch. To accomplish this, promoter induction studies were used to find stronger promoters for the M-PFC, optimization of the riboswitch as a method for inducible protein expression within this system, and the addition of both elements to the existing version of the M-PFC. This M-PFC targets DosR homodimerization; this process is known to be essential for survival within the host. This study optimizes a system that may be used to screen for drugs that are capable of interrupting this interaction.
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Date Issued
2017
Identifier
CFH2000190, ucf:46030
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFH2000190
Computational Methods for Analyzing RNA Folding Landscapes and its Applications
Title
Computational Methods for Analyzing RNA Folding Landscapes and its Applications.
Creator
Li, Yuan, Zhang, Shaojie, Hua, Kien, Jha, Sumit, Hu, Haiyan, Li, Xiaoman, University of Central Florida
Abstract / Description
Non-protein-coding RNAs play critical regulatory roles in cellular life. Many ncRNAs fold into specific structures in order to perform their biological functions. Some of the RNAs, such as riboswitches, can even fold into alternative structural conformations in order to participate in different biological processes. In addition, these RNAs can transit dynamically between different functional structures along folding pathways on their energy landscapes. These alternative functional structures...
Show moreNon-protein-coding RNAs play critical regulatory roles in cellular life. Many ncRNAs fold into specific structures in order to perform their biological functions. Some of the RNAs, such as riboswitches, can even fold into alternative structural conformations in order to participate in different biological processes. In addition, these RNAs can transit dynamically between different functional structures along folding pathways on their energy landscapes. These alternative functional structures are usually energetically favored and are stable in their local energy landscapes. Moreover, conformational transitions between any pair of alternate structures usually involve high energy barriers, such that RNAs can become kinetically trapped by these stable and local optimal structures.We have proposed a suite of computational approaches for analyzing and discovering regulatory RNAs through studying folding pathways, alternative structures and energy landscapes associated with conformational transitions of regulatory RNAs. First, we developed an approach, RNAEAPath, which can predict low-barrier folding pathways between two conformational structures of a single RNA molecule. Using RNAEAPath, we can analyze folding pathways between two functional RNA structures, and therefore study the mechanism behind RNA functional transitions from a thermodynamic perspective. Second, we introduced an approach, RNASLOpt, for finding all the stable and local optimal structures on the energy landscape of a single RNA molecule. We can use the generated stable and local optimal structures to represent the RNA energy landscape in a compact manner. In addition, we applied RNASLOpt to several known riboswitches and predicted their alternate functional structures accurately. Third, we integrated a comparative approach with RNASLOpt, and developed RNAConSLOpt, which can find all the consensus stable and local optimal structuresthat are conserved among a set of homologous regulatory RNAs. We can use RNAConSLOpt to predict alternate functional structures for regulatory RNA families. Finally, we have proposed a pipeline making use of RNAConSLOpt to computationally discover novel riboswitches in bacterial genomes. An application of the proposed pipeline to a set of bacteria in Bacillus genus results in the re-discovery of many known riboswitches, and the detection of several novel putative riboswitch elements.
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Date Issued
2012
Identifier
CFE0004400, ucf:49365
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0004400
Structural and Functional Studies of Glycine Riboswitches and Development of Fab Chaperone Assisted RNA Crystallography
Title
Structural and Functional Studies of Glycine Riboswitches and Development of Fab Chaperone Assisted RNA Crystallography.
Creator
Sherman, Eileen, Ye, Jingdong, Kolpashchikov, Dmitry, Koculi, Eda, Harper, James, Self, William, University of Central Florida
Abstract / Description
The glycine riboswitch is a structured RNA found upstream of genes in mRNA transcripts in many bacteria, functioning as a biofeedback gene regulator. Upon binding glycine, a complete RNA transcript including gene sequences is transcribed, effectively turning on gene expression. In an effort to understand the intricacies of its functioning, many mutants of the riboswitch were made and characterized during Ph. D. work, resulting in discovery of a P0 duplex/kink-turn motif involving a few...
Show moreThe glycine riboswitch is a structured RNA found upstream of genes in mRNA transcripts in many bacteria, functioning as a biofeedback gene regulator. Upon binding glycine, a complete RNA transcript including gene sequences is transcribed, effectively turning on gene expression. In an effort to understand the intricacies of its functioning, many mutants of the riboswitch were made and characterized during Ph. D. work, resulting in discovery of a P0 duplex/kink-turn motif involving a few nucleotides upstream of the established glycine riboswitch sequence which changed its ligand binding characteristics (Chapter 1). Previously, the two aptamers of the riboswitch were thought to cooperatively bind glycine, but with the inclusion of this leader sequence which forms a kink turn motif with the linker between the two aptamers, glycine binding in one aptamer no longer requires glycine binding in the other. Furthermore, the Kd from three species tested are now a similar, lower value of about 5 (&)#181;M, indicating authenticity of this new consensus sequence. Glycine binding and interaptamer interaction both enhanced one another in trans aptamer assays. Another discovery from this was a shortened construct including all of aptamer II but only part of aptamer I in which a few specific nucleotides prevented glycine binding in aptamer II (Chapter 2). This may provide insight into the nature of interaptamer interactions in the full switch; addition of an oligonucleotide complimentary to these nucleotides restored glycine binding ability to aptamer II. With future development, this could also be a useful molecular biology tool, using two signals, glycine and an oligonucleotide, to allow gene expression.To precisely understand how any macromolecule functions, a 3D structure, obtainable by x-ray crystallography, is vital. A new technique to accomplish that for RNA, precedented in the protein world, is Fab chaperoned crystallography, which has advantages compared to RNA alone. A phage displayed library of Fabs with reduced codon diversity designed for RNA was created, the YSGR Min library (Chapter 3). Its Fabs had specificities and affinities equal to or greater than previous libraries which were originally created for phage displayed selection against proteins. Fab chaperoned RNA crystallography is currently in progress for the glycine riboswitch; the best resolution thus far is 5.3 (&)#197; (Chapter 4). In addition to providing molecular insight into its gene regulation mechanism, a structure of the glycine riboswitch could be applied for use in structure based drug design of novel antibiotics targeting the riboswitch to disrupt important downstream carbon cycle genes in pathogenic bacteria.
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Date Issued
2014
Identifier
CFE0005549, ucf:50285
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005549