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Regulation of gene expression by RNA polymerase (RNAP) is an essential ability of living organisms, required for their adaption to a changing environment and ultimately enabling their survival. Interaction of RNAP with ribonucleic acids (DNA or RNA) is crucial for transcription and its regulation. This Doctoral Thesis contains two projects addressing interactions of RNAP with nucleic acids: (i) Transcription of modified DNA templates and (ii) Ms1, a small RNA (sRNA) from M. smegmatis. (i) We investigated the influence of modifications in the major groove of DNA on bacterial transcription in vitro. We found out that transcription of modified DNA templates is influenced on the transcription initiation level and that the promoter sequence is important for the effect of the modifications. Furthermore, we successfully performed transcription switch ON and OFF in vitro by bioorthogonal reactions. This regulation of transcription by artificial DNA modifications has a future in biotechnologies and/or medical therapy. (ii) Regulators of transcription are also small non-coding RNAs. These molecules have an important role in gene expression regulation among prokaryotes and eukaryotes. Ms1 is an sRNA found in mycobacteria. It makes a complex with the RNAP core and it is abundant in stationary phase (in amounts...

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 217-229).

Functional nucleic acids (FNAs), which include both DNA/RNA aptamers and DNA enzymes, have emerged as promising biological recognition elements for biosensors. These species typically require immobilization on or within a solid support, which is usually interfaced to some kind of signal transducer and readout system when use in biosensor. Our group has successfully immobilized several functional nucleic acids in the past, including fluorescence-signalling DNA enzymes, DNA aptamers and RNA aptamers by entrapping them into porous silica or organosilica materials prepared by the sol-gel method using percursors such as sodium silicate (SS), diglyceryl silane (DGS), tetrametylorthosilicate (TMOS) and trimethoxymethysilane (MTMS). While the earlier work established the ability of entrapped FNAs to retain binding and catalytic activity, only limited information was obtained on how different factors affect the performance of immobilized FNAs, and no information was obtained on the effects of aging and storage conditions on FNA performance. The initial objective of this thesis was to employ advanced fluorescence methods to better understand the nature of immobilized DNA and RNA aptamers, and in particular how entrapment in different sol-gel based materials affected FNA performance for detection of small molecule analytes. It was found that the ability of the entrapped aptamer reporters to remain fully hybridized was the most important factor in terms of signalling capability for both DNA and RNA aptamer reporters. It was also observed that more polar materials derived from SS were optimal for both types of aptamer reporters, since these allowed the entrapped aptamers to remain hydridized to their complementary strands and still retain the dynamic motion needed to undergo structure switching, while providing a minimum degree of leaching. The second objective of my research was to develop a paper-based biosensing device incorporating immobilized DNA and RNA aptamers that could be used in the fields of point-of-care diagnostics to further expand the utility of structure-switching aptamer reporters to real world application. A dual response (fluorescence / colorimetric) paper-based sensor utilized printed graphene oxide to immobilize both a RNA and a DNA aptamer in a recognition zone. Upon target addition, the aptamer desorbed and eluted to an amplification zone where rolling circle amplification was used to generate a colorimetric output. This sensor could function with clinical samples such as serum and stool, and allowed detection of key bacterial markers (ATP and glutamate dehydrogenase) at clinically relevant levels. / Thesis / Doctor of Philosophy (PhD)

All biological processes are fundamentally inter-molecular interactions. In order to understand, and hence control, biomolecular structure and function, methods are required that probe biological systems at the molecular level, ideally with those molecules being in their native environment. The research summarized herein has at its core the development and application of ultra violet (UV)-visible spectrophotometric techniquies for this prupose, in particular circular dichrosim (CD) and linear dichrosim (LD) but also absorbance, fluorescence and resonance light scattering. The spectroscopy is complemented by fundamental theoretical work on molecular structure and reactivity that forms the basis for designing molecules to bind to biomolecules for a particular structural or functional effect. A brief summary of the contributions of the listed publications to our understanding of 'Molecular aspects of biololecule structure and function' is given below under five headings: Circular dichroism theory Molecular geometry and reactivity Small molecule-macromolecule interactions: spectroscopic probes of inter-molecular geometries Molecular design for nucleic acid structure and control Spectroscopic probes of biomolecule structure: instrumentation and application In general terms these correspond to successive phases of the research programme, however, all areas have been present since the first publications in 1983 and can be traced weaving through all subsequent activity.

All biological processes are fundamentally inter-molecular interactions. In order to understand, and hence control, biomolecular structure and function, methods are required that probe biological systems at the molecular level, ideally with those molecules being in their native environment. The research summarized herein has at its core the development and application of ultra violet (UV)-visible spectrophotometric techniquies for this prupose, in particular circular dichrosim (CD) and linear dichrosim (LD) but also absorbance, fluorescence and resonance light scattering. The spectroscopy is complemented by fundamental theoretical work on molecular structure and reactivity that forms the basis for designing molecules to bind to biomolecules for a particular structural or functional effect. A brief summary of the contributions of the listed publications to our understanding of 'Molecular aspects of biololecule structure and function' is given below under five headings: Circular dichroism theory Molecular geometry and reactivity Small molecule-macromolecule interactions: spectroscopic probes of inter-molecular geometries Molecular design for nucleic acid structure and control Spectroscopic probes of biomolecule structure: instrumentation and application In general terms these correspond to successive phases of the research programme, however, all areas have been present since the first publications in 1983 and can be traced weaving through all subsequent activity.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [68]-77).

Preventing the spread of blood-borne infectious diseases is vital to improving global health outcomes, particularly for low- and middle-income countries (LMICs). Sensitive and accurate diagnosis of infections is vital to this effort. Nucleic acid amplification tests (NAATs), which amplify pathogen nucleic acids, are gold-standard techniques for detection and quantification of pathogen levels. However, standard NAATs such as polymerase chain reaction (PCR) require expensive equipment for blood sample processing and DNA/RNA amplification, making them challenging to implement in resource-limited areas of LMICs. In this work, I developed two methods to simplify sample processing and amplification to make NAATs more accessible for use at the point of care in resource-limited areas of LMICs. The first method enables instrument-free nucleic acid extraction from whole blood. A room temperature lysis chemistry and a paper-and-plastic sample capture device were developed to isolate, purify, and store pathogen DNA and RNA on a paper capture membrane. Extracted nucleic acids can be eluted and used in standard NAATs or in developmental amplification assays. I demonstrated successful isolation of HIV virion RNA and P. falciparum parasite DNA from whole blood samples over several concentrations with >60% recovery. Extracted RNA remains stable on the capture membrane for two weeks at room temperature and 37°C, alleviating the need for cold storage after sample collection. These results are a promising step toward using this method for simplified sample extraction and storage in low-resource settings in LMICs. The second method I developed is a novel isothermal amplification technique for P. falciparum DNA. Sensitive diagnosis of P. falciparum infection is vital to identify and treat low-density, asymptomatic infections and move closer to eliminating malaria. Highly sensitive PCR assays are difficult to deploy in resource-limited areas of LMICs and existing isothermal methods require complex assay design and are often not sensitive enough to diagnose asymptomatic infections. Here, I developed a novel isothermal technique which amplifies multiple regions of the P. falciparum genome, generating a large amount of DNA for better analytical sensitivity. The assay achieves a lower limit of detection of ~23.4 fg P. falciparum gDNA/µL (~1 parasite/µL) in 30 minutes, similar gold-standard PCR assay while using a fraction of the resources required for PCR. Lastly, I adapted the assay for implementation at the point of care. I showed that the assay directly amplifies P. falciparum parasite DNA captured on paper with the paper-and-plastic device previously developed. I also incorporated visual assay readout with lateral flow strips, eliminating the need for specialized equipment to detect amplified DNA. I explored methods to eliminate cold storage of reagents by stabilizing amplification enzymes at room temperature. The work described in this thesis represents two enhanced methods for point of care detection of blood borne pathogens. By simplifying sample extraction, amplification, and detection, the methods described here make NAATs more accessible to low-resource areas of LMICs. The whole blood nucleic acid extraction device and isothermal assay described in this work can be used together for sensitive diagnosis of P. falciparum malaria. The methods can also be used independently, or in combination with other techniques routinely used in the field. The flexibility built in to these methods enables easier integration into existing workflows in LMICs. / 2021-05-18T00:00:00Z

Motivation: Understanding how nucleic acids interact is essential for understanding their function. Controlling these interactions, for example, can allow us to detect diseases and create new therapeutics. During quantitative reverse-transcription polymerase chain reaction (qRT-PCR) testing, having nucleic acids interact as designed is essential for ensuring accurate test results. Accurate testing is an important consideration during the detection of COVID-19, the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Results: I introduced the program DinoKnot (Duplex Interaction of Nucleic acids with pseudoKnots) that follows the hierarchical folding hypothesis to predict the secondary structure of two interacting nucleic acid strands (DNA/RNA) of similar or different type. DinoKnot is the first program that utilizes stable stems in both strands as a guide to find the structure of their interaction. Using DinoKnot, I predicted the interaction structure between the SARS-CoV-2 genome and nine reverse primers from qRT-PCR primer-probe sets. I compared these results to an existing tool RNAcofold and highlighted an example to showcase DinoKnot’s ability to predict pseudoknotted structures. I investigated how mutations to the SARS-CoV-2 genome may affect the primer interaction and predicted three mutations that may prevent primer binding, reducing the ability for SARS-CoV-2 detection. Interaction structure results pre- dicted by DinoKnot that showed disruption of primer binding were consistent with a clinical example showing detection issues due to mutations. DinoKnot has the potential to screen new SARS-CoV-2 variants for possible detection issues and support existing applications involving DNA/RNA interactions, such as microRNA (miRNA) target site prediction, by adding structural considerations to the interaction to elicit functional information. / Graduate

<p> The purpose of this research is to develop a general method for capturing and separating specific targets. Nucleic acid aptamers are short sequences of single-stranded DNA or RNA which have the ability to bind to the small organic or inorganic molecules such as protein and metal ions with high binding affinity. In this study, a bioconjugate re-usable system was developed. It can reversibly load or unload DNA aptamers.</p> <p> To allow separation, a thermally responsive polymer (N-isopropylacrylamide, PNIPAM) is used. This polymer can undergo a reversible phase transition upon adding NaCl and/or increasing temperature. A short sequence of single stranded DNA (ssDNA) was coupled to PNIPAM. The ssDNA will experience a reversible phase transition because of the PNIPAM.</p> <p> The DNA sequence for an aptamer can be extended to contain a sequence that is complementary to that of the ssDNA coupled to PNIPAM. Adding this extended aptamer to the conjugate will result in spontaneous hybridization of the two strands of DNA. These strands can be separated using an agent (e.g. urea) that destroys hydrogen bonding. The conjugate can be recovered using a reversible inverse phase transition.</p> <p> The same PNIPAM-ssDNA conjugate can be used reversibly for coupling different aptamers. The aptamers did not lose their binding ability when coupled with PNIPAM-ssDNA conjugates. In the process of precipitation separation targets, the PNIPAM-ssDNA conjugate showed little loss with applied phase transition. Moreover, the coupling efficiency of the ssDNA to PNIPAM conjugates was determined. The binding ability of the ATP aptamers to ATP was also investigated.</p> / Thesis / Master of Applied Science (MASc)