title: High-Throughput Sequencing Based Probing of Protein/RNA Structures and Functions
Creators: name: Solayman, Md.
Publisher: name: Griffith University
Year: 2022
Subjects: subject: high-throughput sequencing (HTS)
subject: RNA
subject: Single molecule real time (SMRT)
subject: DNA sequencing

description: The rapid advancement in sequencing chemistry, sequencing technologies, and bioinformatics has significantly increased the sequencing automation and lowered the cost. The applications of high-throughput sequencing (HTS) technologies are expanding from research laboratories to diagnostic clinics on a regular basis. Moreover, diverse methods used in epigenetics, proteomics, structure probing of macromolecules (DNA, RNA, and proteins) have been developed based on the HTS technology. This thesis describes the development of two novel techniques, high-throughput split-protein profiling (HiTS) and RNA solvent accessibility probing method (RL-Seq), broadening the applications of HTS technologies for probing protein/RNA structures and functions. Chapter 1 of the thesis provides an overview of the history of HTS technologies, available platforms, ongoing development in this field, and their diverse applications, particularly in the area of proteomics and RNA structure probing. In Chapter 2, we introduced the HiTS method that allowed fast identification of self- and assisted complementary positions of three antibiotic-resistant proteins (fosfomycin, fosA3; erythromycin, ermB; and chloramphenicol, catI resistant-proteins). The finding of suitable split sites in proteins is important because they are used as reporters in protein complementary assay (PCA) for studying protein-protein interactions in different organisms. However, only a small number of split-protein systems have been identified so far owing to manual, labourintensive optimization of the candidate genes. The proposed HiTS method employs transposon mutagenesis, conditional interaction of split fragments by rapamycin-regulated FRB-FKBP protein pairs, and deep sequencing for fast identification of self- and assisted complementary fragments, which are subsequently confirmed by low-throughput testing. In Chapter 3, we further applied the HiTS method on T7 RNA polymerase (T7 RNAP), a bacteriophage RNA polymerase, considering its importance in synthetic biology in addition to the PCA. We found that the newly developed HiTS method could also be applicable to T7 RNAP for locating suitable split sites for self-complementing variants. Several selfcomplementing variants were found and one with a stronger signal than the wild type one. In Chapter 4, in preparation of applying HTS technology to probe RNA solvent accessibility, we reviewed the available experimental and computational techniques for RNA solvent accessibility studies and identified existing research gaps. Current experimental approaches for studying RNA solvent accessibility include hydroxyl radical probing (HRF-Seq), light activated structural examination of RNA (LASER), and its modified versions (LASER-Seq, LASER-Map, and icLASER). The reactivity readouts of these methods are based on either the reverse transcriptase stop (RT-stop) at cleavage points or mutational profiling at adduct formation sites. These approaches rely on reverse transcriptase enzymes and random primers, which suffer from non-specific drop-off to create short truncated sequences, which successively lead to false-positive signals at probe-reactive sites. In Chapter 5, we proposed the RL-Seq (RtcB Ligation-Seq) method to overcome the abovementioned limitations of the existing approaches. The method is illustrated by measuring the solvent accessibility of Escherichia coli complete ribosomal complexes at the single-nucleotide resolution. In this method, unique properties of RtcB ligase were used to identify the probing sites by ligating a pre-defined 5′-OH end containing linker with the hydroxyl radicals cleavage generated 3′-P ends. The application of this method to ribosomal RNAs (23S, 16S, and 5S rRNAs) confirmed its ability to estimate solvent accessibility with high sensitivity (required low sequencing depth) and accuracy (strong correlation to structure-derived values). In addition, the pre-defined linker employed in this method allowed using of a fixed primer in reverse transcription reaction and significantly minimized the biases during subsequent PCR amplification. In Chapter 6, we discussed the future prospects of these HTS technology-based methods developed in this thesis.