Date of Award

1-1-2024

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Chemistry

Dissertation/Thesis Chair

Jia Sheng

Committee Members

Li Niu, Gabriele Fuchs, Maksim Royzen

Keywords

CRISPR-Cas System, G-quadruplexes, Inosine, Mass spectrometry, Phosphorothioate, RNA Modifications

Subject Categories

Biochemistry

Abstract

Nucleic acid modifications have gained compelling interest for their pivotal roles in cellular and biological processes. These modifications introduced a new layer of regulation without altering the original genetic sequence. In this burgeoning field of epitranscriptomics, many of the chemical modifications have emerged as potential biomarkers in therapeutic applications. Inosine is an essential post-transcriptional RNA modification that has functional roles in modulating the fate of all RNAs. The enzyme ADAR catalyzes the deamination of adenosine to inosine. This can also lead to change in the eukaryotic transcriptome due to A-G miscoding since inosine preferentially base pairs with cytosine. Moreover, hyper-editing or multiple A to G transitions in clusters were detected in measles virus (MV). Inosine modifications either directly on viral RNA or on cellular RNA can have anti or pro-viral repercussions. While many of the significant roles of inosine in cellular RNAs are well understood, the effects of hyper-editing of A to I on viral polymerase activity during RNA replication remain elusive. Moreover, biological strategies such as molecular cloning and RNA-seq for transcriptomic interrogation rely on RT-PCR with little to no emphasis placed on the first step reverse transcription, which may reshape the sequence results when hyper-modification is present. In chapter 2, we systematically explore the influence of inosine modification, varying the number and position of inosines, on decoding outcomes, using three different reverse transcriptases (RTs) followed by standard Sanger sequencing. We find that inosine alone or in clusters can differentially affect RT activity. To gain structural insights into the accommodation of inosine in the polymerase site of HIV-1-RT and how this structural context affects the base pairing rules for inosine, we perform molecular dynamics simulations of the HIV-1 reverse transcriptase (HIV-1-RT). The simulations highlight the importance of the protein-nucleotide interaction as a critical factor when deciphering the base pairing behavior of inosine clusters. This effort sets the groundwork for decrypting the physiological significance of inosine and linking the fidelity of reverse transcriptase and the possible diverse transcription outcomes of cellular RNAs and/or viral RNAs where hyper-edited inosines are present in the transcripts. Deoxyinosine is generated from the deamination of deoxyadenosine from exposure to nitrosative compounds in the environments. Due to the lack of N2-amino group and the similar electrostatic potentials of the two, the G-to-I mutation was commonly used to study inosine induced Hoogsteen base paring, as well as the effects of ligand binding to both duplex and quadruplex DNAs. The telomeric DNA, a distal region of eukaryotic chromosome containing guanine-rich repetitive sequence of (TTAGGG)n, which can adopt G-quadruplexes (G4s). Previous studies have demonstrated the implication of G4 in tumor inhibition. In chapter 3, we investigated how the structural dynamics of a G-quadruplex, formed by the human telomeric sequence is affected by inosine, a prevalent modified nucleotide. We used the standard (TTAGGG)n telomere repeats with guanosine mutated to inosine at each G position. Sequences (GGG)4, (IGG)4, (GIG)4, (GGI)4, (IGI)4, (IIG)4, (GII)4, and (III)4, bridged by TTA linker, are studied using biophysical experiments and molecular modeling. The effects of metal cations in quadruplex folding were explored in both Na+ and K+ containing buffers using CD and UV-melting studies. Our results show that antiparallel quadruplex topology forms with the native sequence (GGG)4 and the terminal modified DNAs (IGG)4 and (GGI)4 in both Na+ and K+ containing buffers. Specifically, quadruplex hybrid was observed for (GGG)4 in K+ buffer. Among the other modified sequences, (GIG)4, (IGI)4 and (GII)4 show parallel features, while (IIG)4 and (III)4 show no detectable conformation in the presence of either Na+ or K+. Our studies indicate that terminal lesions (IGG)4 and (GGI)4 may induce certain unknown conformations. The folding dynamics become undetectable in the presence of more than one inosine substitution except (IGI)4 in both buffer ions. In addition, both UV melting and CD melting studies implied that in most cases the K+ cation confers more thermodynamic stability compared to Na+. Collectively, our conformational studies revealed the diverse structural polymorphisms of G4 with positional dependent G-to-I mutations in different ion conditions. Beyond the nucleobase modifications found in DNA and RNA, the discovery of phosphorotioate (PS) on DNA such that one of the non-bridging oxygens is replaced by the sulfur in the sugar-phosphate backbone has again amazed us on this ingeniously evolved protective mechanism against nuclease degradation in bacteria. Since then, scientists have extended the application of this physiological DNA modification on gene therapeutics to reduce immune response and enhance transcription/translation. Due to the architectural similarity of DNA and RNA, we were wondering about the existence of RNA phosphorotioate (PS) modification. In chapter 4, we set out a method for both detection and quantification of RNA PS modification in a serious model cells using mass spectrometry. We reported the existence of PS modification in both eukaryotes and prokaryotes. The GpsG modification exists in the Rp configuration and was quantified with liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). By knocking out the DndA gene in E. coli, we show the Dnd clusters that regulate DNA PS modification may also play roles in RNA PS modification. We also show that the GpsG modification locates on rRNA in E. coli, L. lactis, and HeLa cells, and it is not detected in rRNA-depleted total RNAs from these cells. The aforementioned PS modification naturally occurred in the genome of bacteria as restriction-modification (R-M) system against phage invasion. CRISPR-Cas system is another mechanism possesses by the bacteria as an adaptive immunity against foreign DNA interference. This programmable DNA editing tool has shown promising applications in treating human genetic diseases. Clinical success of CRISPR technology is dependent on incorporation of modifications into the single guide RNA (sgRNA). However, chemical synthesis of modified sgRNAs, which are over 100 nucleotides in length, is difficult and low-yielding. In chapter 5, we developed a conjugation strategy that utilized bio-orthogonal chemistry to efficiently assemble functional sgRNAs containing nucleobase modifications. The described approach entails the chemical synthesis of two shorter RNA oligonucleotides: a 31-mer containing tetrazine (Tz) group and a 70-mer modified with a trans-cyclooctene (TCO) moiety. The two oligonucleotides were conjugated to form functional sgRNAs. The two-component conjugation methodology was utilized to synthesize a library of sgRNAs containing nucleobase modifications such as m1A, m6A, s2U and s4U. The impacts of these RNA modifications on overall CRISPR activity were investigated in vitro and in Cas9-expressing HEK293T cells. Our study demonstrated that the nature and the position of modification can have drastically different impacts on the CRISPR editing outcomes. In addition to CRISPR-Cas9 for DNA editing, CRISPR-Cas13 also holds promise to correct gene causing diseases at the transcripts level. Discovered in recent years, Cas13 is a single effector CRISPR associated RNA-guided RNase that can be programmed to cleave desire single stranded RNA to induce RNA decay. Inactive or dead Cas13 (dCas13) can be engineered to precisely locate target RNA sequences without endonuclease activity, this way when fuse with other functional moiety such as effector enzyme (reader, writer or eraser) can achieve regulation at local transcript level. In chapter 6, we have engineered fusion constructs with dCas13b fused with Nsun2 (m5C writer) and separately fused with TET (m5C eraser) to study the potential role of m5C on mRNA. This is still an ongoing project with data presented here are unpublished results.

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