Browsing by Author "Sczepanski, Jonathan"

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CLAP-seq: An Aptamer-Based Platform for Transcriptome-Wide Mapping of RNA Modifications
Chemistry; TAMU; https://hdl.handle.net/20.500.14641/200; DHHS-NIH-Eunice Kennedy Shriver National Institute of Child Health & Human Development
Project Summary/Abstract Beginning in the 1950s, more than 100 types of posttranscriptional modifications have been identified in cellular RNA. Today, the study of RNA post-transcriptional modifications – known as epitranscriptomics – is a rapidly developing field, which promises to greatly enhance our understanding of human health and disease. Despite the profound implications already assigned to many RNA modifications, their precise functions remain poorly understood. This can be attributed to the lack of sensitive and robust sequencing technologies to detect these epitranscriptomics marks in a transcriptome-wide manner. A key bottleneck is the lack of sensitive and specific enrichment techniques (affinity- or reactivity-based) for RNA molecules containing these modifications. The proposed research takes direct aim at this critical deficit using the aptamer approach, employing in vitro selection methods to identify nucleic acid molecules that bind chemically modified RNAs. These aptamers are unique in that they are comprised of L-(deoxy)ribose-based nucleic acids (L-DNA and L-RNA), which are mirror images (enantiomers) of natural D-nucleotides. L-Aptamers, which are completely orthogonal to natural biology, are extremely well suited for binding RNA targets. Therefore, in vitro selection will be used to isolate novel L- aptamers capable of binding chemically modified mononucleotides, which will enable selective capture of RNA molecules containing the same modified residue. These L-aptamers will then be used in Cross-Linking- Aptamer Pull-down and sequencing (CLAP-seq), the first transcriptome-wide profiling technology employing aptamer-based RNA enrichment prior to next-generation sequencing. CLAP-seq not only promises to open a general and robust route towards transcriptome-wide profiling of the growing list of RNA modifications, but also promises to reinforce our current view of the epitranscriptome. Accordingly, the development of CLAP-seq will have a profound impact on the field of epitranscriptomics, which is well aligned with the mission of the NICHD and the goal of this FOA: to promote research into the role of RNA chemical modifications in development and related disease.
Mirror Image Aptamers: Next Generation RNA-Binding Reagents for Basic Research and Therapeutic Applications
Chemistry; TAMU; https://hdl.handle.net/20.500.14641/200; DHHS-NIH-National Institute of General Medical Science
Project Summary/Abstract The increasing appreciation of RNA's structure-function relationship has led to a demand for new technologies that enable targeting of specific RNA structures. Such technologies are essential for the development of probes to study RNA function and therapeutics to treat RNA-mediated diseases. However, outside of antibiotics binding the ribosome, structure-specific RNA-binding reagents are very rare. Thus, developing of new technologies that enable structure-specific targeting of RNA remains an important challenge in many fields. The central vision of my research program is to address the deficit of structure-specific RNA-binding reagents using a radically different type of nucleic acid affinity reagent: L-aptamers. L-Aptamers are unique because they are comprised of L-(deoxy)ribose-based nucleic acids (L-DNA and L-RNA), which are mirror images (enantiomers) of natural D-nucleotides. Because oligonucleotides of opposite stereochemistry (D versus L) are incapable of forming contiguous Watson-Crick base pairs with each other, we are able to evolve L-aptamers that adaptively bind structured D-RNA targets through tertiary interactions (shape) rather than primary sequence. In other words, L-aptamers escape the tyranny of Watson-Crick base pairing, enabling a more nuanced mode of molecular recognition to be discovered. As a result, L-aptamers bind structured RNAs with greater affinity and specificity compared to conventional affinity reagent. Binding RNAs based on their shape rather than Watson-Crick base pairing represents a significant departure from traditional oligonucleotide-based approaches and represents a major advance in aptamer technology. During the next five year, my research group aims to further develop L-aptamer technology in order to realize its promise as a practical research and therapeutic tool. In particular, we will focus on incorporation of modified nucleotides that bestow protein-like functionality on L-aptamers, thus generating a novel class of RNA-targeted antibody mimetics. Because these technological developments will be carried out in the context of disease associated RNAs, such as oncogenic microRNAs and viral RNAs, this work will have an immediate impact by generating lead reagents to probe the etiology of disease and develop new therapeutic strategies. In line with my vision, we aim to determine the structure of an L-aptamer–D-RNA complex, which will provide insight into this novel mode of recognition and inform future L-aptamer design.
Mirror Image Aptamers: Next Generation RNA-Binding Reagents for Basic Research and Therapeutic Applications
Chemistry; TAMU; https://hdl.handle.net/20.500.14641/200; DHHS-NIH-National Institute of General Medical Science
The primary focus of this research proposal will be the identification, discovery, and elucidation of novel biochemical pathways for the metabolism of complex carbohydrates in the human gut microbiome. Currently, more than one thousand different bacterial species have been identified in the human intestinal tract and the total number of genes contained within these bacteria exceeds the number of human genes by more than two orders of magnitude. Moreover, it has been demonstrated that the composition of the human gut microbiome and the associated metabolic diversity contained within these bacteria contribute significantly to the maintenance of human health and physiology. Unfortunately, a significant fraction of the enzymes and corresponding metabolic pathways contained within the bacteria found in the human gut have an uncertain, unknown, or incorrect functional annotation. This uncertainty suggests that a substantial fraction of the metabolic potential contained within the human gut microbiome remains to be properly characterized. Our proposed experimental approach for the discovery and elucidation of novel metabolic pathways for the metabolism of complex carbohydrates will involve the concerted and synergistic utilization of bioinformatics, computational biology, three-dimensional protein structure determination, metabolomics and physical screening of focused compound libraries. The determination of the substrate and reaction diversity contained within the newly discovered enzyme-catalyzed reactions will provide unique insights into the molecular mechanisms for the evolution and development of novel enzymatic activities and will provide potential targets for therapeutic intervention.
Mirror image DNA circuitry for complex microRNA analysis in live cells
Chemistry; TAMU; https://hdl.handle.net/20.500.14641/200; DHHS-NIH-National Institute of BioMedical Imaging and BioEngineering
Project Summary/Abstract MicroRNAs (miRNAs) are short, noncoding RNAs that play a critical role in post-transcriptional regulation of gene expression. Consequently, aberrant miRNA expression levels are associated with a wide range of human diseases, the most prominent of which is cancer. Cancer cells are often associated with the simultaneous up- and-down regulation of several miRNAs relative to normal cells of the same tissue, and these “signatures” can reveal significant information about the underlying disease. Thus, nucleic acid analysis technologies aimed at profiling miRNA expression patterns in living cells and tissues hold great promise for early cancer detection and diagnosis. However, prevailing methods for detecting nucleic acids in living systems are generally limited to the detection of a single nucleic acid target, thereby precluding their use in more complex pattern- recognition applications. A promising solution to this problem is molecular circuitry, and specifically, DNA strand-displacement circuits that can be programmed to generate optical and/or chemical “outputs” in response to specific combinations of nucleic acid “inputs”. Unfortunately, exogenously delivered DNA has a cellular half- life on the order of minutes and is susceptible to unintended interactions with cellular macromolecules, all of which adversely affect the performance of the device. As a consequence, it remains enormously challenging to execute complex and useful tasks in living systems using DNA-based circuits. Herein, the PI provides an innovative solution to this problem: mirror image DNA. Mirror image DNA (referred to as L-DNA) has the same physical and chemical properties as its natural counterpart, D-DNA, yet as a reflection, it is completely invisible to the stereospecific environment of cells (i.e. L-DNA is resistant to both nuclease degradation and off-target interactions with cellular components). Consequently, DNA-based circuitry constructed from L-DNA is expected to operate free from cellular interference, thereby overcoming the primary barrier to engineering complex and reliable functionality. On this basis, the PI proposes to develop a series of autonomous L-DNA-based circuits capable of recognizing and reporting specific miRNA expression patterns in live human cells. The immediate goal of this work is to uncover fundamental relationships between circuit design and cellular delivery methods, which together represent a critical first step towards constructing more complex intracellular devices. As the project progresses, the complexity of the L-DNA circuitry will be gradually increased through the introduction of L-DNA-based logic gate motifs that will be arranged to compute the presence of specific combinations of up to four different miRNAs. If successful, this work will signify a major advance in the area of intracellular DNA computing and provide a strong foundation for future applications aimed at “intelligent” disease diagnosis.