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Base editors are tools that chemically modify the nucleobases of DNA and RNA in a programmable manner, allowing for genome, epigenome, and transcriptome editing in live cells. These tools can be used to introduce specific base transitions in DNA or RNA, manipulate methylation patterns in the epigenome, and create genetically encoded libraries in target genes. These various functions can be used to modulate every aspect of the central dogma. The efficiency and precision of base editors makes them useful in both basic research and in the development of new therapies. The adenosine base editor (ABE) was developed by evolving the RNA-modifying enzyme TadA to also accept DNA as substrate. This suggests that the base editing tool kit can be expanded to use many of the naturally occurring RNA-modifications as intermediates. However, current base editors remain limited to deaminases, suggesting that more complex chemical reactions face additional barriers. To better inform the creation of new base editors, we set out to better understand the molecular mechanisms of the wild-type TadA enzyme (used in ABE0.1) and the DNA-modifying variants developed for base editing (ABE7.10, ABE8e, ABE8.20, etc.). First, we observe that ABE0.1 shows high Cas9-independent, off-target RNA-editing at UACG motifs in accessible loops. In chapter 3, we ask if this discovery also extends to DNA and find that ABE0.1 can perform targeted DNA-editing at TACG motifs. This editing is inefficient though and may be easily missed, so we leveraged this discovery to develop a fluorescent reporter of base editing that greatly increases our ability to detect low-levels of base editing. We finish out the chapter by using this fluorescent reporter to better understand the sequence-dependent editing of ABE0.1 and evolved ABEs. Finally, we apply this new understanding of an early base editor to develop more sequence-optimized fluorescent reporters to: detect DNA editing by RNA-modifying enzymes that could be used

Biosensors are analytical devices that utilize biological sensing elements, such as enzymes, antibodies, nucleic acids, or cells, to detect a given analyte [...].

137 p (eusk.) 130 p. (eng.), La enfermedad celíaca es una enfermedad inflamatoria crónica desarrollada en individuos con susceptibilidad genética por una respuesta inmune inadecuada al gluten de la dieta. Sin embargo, la respuesta inmunitaria inducida por el gluten derivada de la susceptibilidad genética explica menos del 50% de la heredabilidad, lo que sugiere que además del gluten podría haber factores ambientales adicionales que contribuyan al inicio de la respuesta inmunitaria. En base a esto, se han propuesto las infecciones virales como posibles agentes desencadenantes en la enfermedad celiaca.Con el fin de investigar la implicación de las infecciones virales en el desarrollo de la enfermedad celiaca, el proyecto actual se ha centrado en la búsqueda de determinantes funcionales relacionados con la respuesta inmunitaria innata contra las infecciones por virus de ARN. Más concretamente, en la presente tesis doctoral se ha analizado una nueva capa de regulación génica de IRF7 tras la combinación de infecciones virales entéricas y el consumo de gluten, y se ha estudiado la implicación de la metilación del ARN m6A en la regulación de este transcrito antiviral y su actividad aguas abajo en la vía de señalización del IFN-I.

Living cells are extremely complicated systems and composed of hundreds of thousands of diverse biomolecules, such as proteins, nucleic acids, and carbohydrates [...].

Multiple sclerosis is a chronic inflammatory disease of the central nervous system1. Astrocytes are heterogeneous glial cells that are resident in the central nervous system and participate in the pathogenesis of multiple sclerosis and its model experimental autoimmune encephalomyelitis2,3. However, few unique surface markers are available for the isolation of astrocyte subsets, preventing their analysis and the identification of candidate therapeutic targets; these limitations are further amplified by the rarity of pathogenic astrocytes. Here, to address these challenges, we developed focused interrogation of cells by nucleic acid detection and sequencing (FIND-seq), a high-throughput microfluidic cytometry method that combines encapsulation of cells in droplets, PCR-based detection of target nucleic acids and droplet sorting to enable in-depth transcriptomic analyses of cells of interest at single-cell resolution. We applied FIND-seq to study the regulation of astrocytes characterized by the splicing-driven activation of the transcription factor XBP1, which promotes disease pathology in multiple sclerosis and experimental autoimmune encephalomyelitis4. Using FIND-seq in combination with conditional-knockout mice, in vivo CRISPR-Cas9-driven genetic perturbation studies and bulk and single-cell RNA sequencing analyses of samples from mouse experimental autoimmune encephalomyelitis and humans with multiple sclerosis, we identified a new role for the nuclear receptor NR3C2 and its corepressor NCOR2 in limiting XBP1-driven pathogenic astrocyte responses. In summary, we used FIND-seq to identify a therapeutically targetable mechanism that limits XBP1-driven pathogenic astrocyte responses. FIND-seq enables the investigation of previously inaccessible cells, including rare cell subsets defined by unique gene expression signatures or other nucleic acid markers.

Extracellular vesicles (EVs) carry diverse bioactive components including nucleic acids, proteins, lipids and metabolites that play versatile roles in intercellular and interorgan communication. The capability to modulate their stability, tissue-specific targeting and cargo render EVs as promising nanotherapeutics for treating heart, lung, blood and sleep (HLBS) diseases. However, current limitations in large-scale manufacturing of therapeutic-grade EVs, and knowledge gaps in EV biogenesis and heterogeneity pose significant challenges in their clinical application as diagnostics or therapeutics for HLBS diseases. To address these challenges, a strategic workshop with multidisciplinary experts in EV biology and U.S. Food and Drug Administration (USFDA) officials was convened by the National Heart, Lung and Blood Institute. The presentations and discussions were focused on summarizing the current state of science and technology for engineering therapeutic EVs for HLBS diseases, identifying critical knowledge gaps and regulatory challenges and suggesting potential solutions to promulgate translation of therapeutic EVs to the clinic. Benchmarks to meet the critical quality attributes set by the USFDA for other cell-based therapeutics were discussed. Development of novel strategies and approaches for scaling-up EV production and the quality control/quality analysis (QC/QA) of EV-based therapeutics were recognized as the necessary milestones for future investigations.

Differential host responses in coronavirus disease 2019 (COVID-19) and multisystem inflammatory syndrome in children (MIS-C) remain poorly characterized. Here, we use next-generation sequencing to longitudinally analyze blood samples from pediatric patients with COVID-19 or MIS-C across three hospitals. Profiling of plasma cell-free nucleic acids uncovers distinct signatures of cell injury and death between COVID-19 and MIS-C, with increased multiorgan involvement in MIS-C encompassing diverse cell types, including endothelial and neuronal cells, and an enrichment of pyroptosis-related genes. Whole-blood RNA profiling reveals upregulation of similar pro-inflammatory pathways in COVID-19 and MIS-C but also MIS-C-specific downregulation of T cell-associated pathways. Profiling of plasma cell-free RNA and whole-blood RNA in paired samples yields different but complementary signatures for each disease state. Our work provides a systems-level view of immune responses and tissue damage in COVID-19 and MIS-C and informs future development of new disease biomarkers.

Base editors are genome editing tools that enable site-specific base conversions through the chemical modification of nucleobases in DNA. Adenine base editors (ABEs) convert A ⋅ T to G ⋅ C base pairs in DNA by using an adenosine deaminase enzyme to modify target adenosines to inosine intermediates. Due to the lack of a naturally occurring adenosine deaminase that can modify DNA, ABEs were evolved from a tRNA-deaminating enzyme, TadA. Previous experiments with an ABE comprising a wild-type (wt) TadA showed no detectable activity on DNA, and directed evolution was therefore required to enable this enzyme to accept DNA as a substrate. Here we show that wtTadA can perform base editing in DNA in both bacterial and mammalian cells, with a strict sequence motif requirement of TAC. We leveraged this discovery to optimize a reporter assay to detect base editing levels as low as 0.01 %. Finally, we used this assay along with molecular dynamics simulations of full ABE:DNA complexes to better understand how the sequence recognition of mutant TadA variants change as they accumulate mutations to better edit DNA substrates.

Environmental omics can provide informative biomarkers for identifying pathologies or physiological responses to environmental change. Nucleic acid adductomics, one of the newest omics techniques, is particularly well suited for assessing exposure and effects of environmental contaminants because the technique can capture modifications at the (epi)genome level. However, identifying potential adduct-biomarkers is challenging, especially in non-model organisms. Therefore, the primary aims of this study were to improve the methodology and explore the applicability of nucleic acid adductomics in environmental monitoring of the biological effects of contaminants, using amphipods as sentinel species. From a methodology perspective, this thesis presents a gradual progression in screening and detecting DNA modifications. The mass spectrometry (MS) based approach for identifying candidate DNA adducts utilized the characteristic neutral loss of deoxyribose. In Paper I, the data generated from high resolution MS in Full Scan-Data independent Acquisition mode were analyzed manually using the fragmentation pattern of nucleoside adduct ions. In Paper II, processing time and efficiency were significantly improved by the development of nLossFinder software. Further, TraceFinder software was combined and used to evaluate and quantify detected adducts (Paper III). Another important development was a simultaneous approach for analysis of both DNA and RNA adducts in a single MS run presented in Paper IV. The analysis of amphipod samples based on the existing approaches and methods developed in this thesis showed that reproductive pathologies identified by the microscopic analysis were associated with specific DNA modifications that can be used to classify field-collected individuals according to their health status (Paper I). Moreover, epigenetic marks were the most influential adducts for this classification. Finally, the adduct profile of amphipods differed between the areas with r

Environmental omics can provide informative biomarkers for identifying pathologies or physiological responses to environmental change. Nucleic acid adductomics, one of the newest omics techniques, is particularly well suited for assessing exposure and effects of environmental contaminants because the technique can capture modifications at the (epi)genome level. However, identifying potential adduct-biomarkers is challenging, especially in non-model organisms. Therefore, the primary aims of this study were to improve the methodology and explore the applicability of nucleic acid adductomics in environmental monitoring of the biological effects of contaminants, using amphipods as sentinel species. From a methodology perspective, this thesis presents a gradual progression in screening and detecting DNA modifications. The mass spectrometry (MS) based approach for identifying candidate DNA adducts utilized the characteristic neutral loss of deoxyribose. In Paper I, the data generated from high resolution MS in Full Scan-Data independent Acquisition mode were analyzed manually using the fragmentation pattern of nucleoside adduct ions. In Paper II, processing time and efficiency were significantly improved by the development of nLossFinder software. Further, TraceFinder software was combined and used to evaluate and quantify detected adducts (Paper III). Another important development was a simultaneous approach for analysis of both DNA and RNA adducts in a single MS run presented in Paper IV. The analysis of amphipod samples based on the existing approaches and methods developed in this thesis showed that reproductive pathologies identified by the microscopic analysis were associated with specific DNA modifications that can be used to classify field-collected individuals according to their health status (Paper I). Moreover, epigenetic marks were the most influential adducts for this classification. Finally, the adduct profile of amphipods differed between the areas with r

Modifications of nucleic acids (DNA and RNA) from oxidative stress is a potential driver of aging per se and of mortality in age-associated medical disorders such as type 2 diabetes (T2D). In a human cohort, we found a strong prediction of all-cause mortality by a marker of systemic oxidation of RNA in patients with T2D (n = 2672) and in nondiabetic control subjects (n = 4079). The finding persisted after the adjustment of established modifiers of oxidative stress (including BMI, smoking, and glycated hemoglobin). In contrast, systemic levels of DNA damage from oxidation, which traditionally has been causally linked to both T2D and aging, failed to predict mortality. Strikingly, these findings were subsequently replicated in an independent general population study (n = 3649). The data demonstrate a specific importance of RNA damage from oxidation in T2D and general aging., Modifications of nucleic acids (DNA and RNA) from oxidative stress is a potential driver of aging per se and of mortality in age-associated medical disorders such as type 2 diabetes (T2D). In a human cohort, we found a strong prediction of all-cause mortality by a marker of systemic oxidation of RNA in patients with T2D (n = 2672) and in nondiabetic control subjects (n = 4079). The finding persisted after the adjustment of established modifiers of oxidative stress (including BMI, smoking, and glycated hemoglobin). In contrast, systemic levels of DNA damage from oxidation, which traditionally has been causally linked to both T2D and aging, failed to predict mortality. Strikingly, these findings were subsequently replicated in an independent general population study (n = 3649). The data demonstrate a specific importance of RNA damage from oxidation in T2D and general aging.

Environmental omics can provide informative biomarkers for identifying pathologies or physiological responses to environmental change. Nucleic acid adductomics, one of the newest omics techniques, is particularly well suited for assessing exposure and effects of environmental contaminants because the technique can capture modifications at the (epi)genome level. However, identifying potential adduct-biomarkers is challenging, especially in non-model organisms. Therefore, the primary aims of this study were to improve the methodology and explore the applicability of nucleic acid adductomics in environmental monitoring of the biological effects of contaminants, using amphipods as sentinel species. From a methodology perspective, this thesis presents a gradual progression in screening and detecting DNA modifications. The mass spectrometry (MS) based approach for identifying candidate DNA adducts utilized the characteristic neutral loss of deoxyribose. In Paper I, the data generated from high resolution MS in Full Scan-Data independent Acquisition mode were analyzed manually using the fragmentation pattern of nucleoside adduct ions. In Paper II, processing time and efficiency were significantly improved by the development of nLossFinder software. Further, TraceFinder software was combined and used to evaluate and quantify detected adducts (Paper III). Another important development was a simultaneous approach for analysis of both DNA and RNA adducts in a single MS run presented in Paper IV. The analysis of amphipod samples based on the existing approaches and methods developed in this thesis showed that reproductive pathologies identified by the microscopic analysis were associated with specific DNA modifications that can be used to classify field-collected individuals according to their health status (Paper I). Moreover, epigenetic marks were the most influential adducts for this classification. Finally, the adduct profile of amphipods differed between the areas with r

Environmental omics can provide informative biomarkers for identifying pathologies or physiological responses to environmental change. Nucleic acid adductomics, one of the newest omics techniques, is particularly well suited for assessing exposure and effects of environmental contaminants because the technique can capture modifications at the (epi)genome level. However, identifying potential adduct-biomarkers is challenging, especially in non-model organisms. Therefore, the primary aims of this study were to improve the methodology and explore the applicability of nucleic acid adductomics in environmental monitoring of the biological effects of contaminants, using amphipods as sentinel species. From a methodology perspective, this thesis presents a gradual progression in screening and detecting DNA modifications. The mass spectrometry (MS) based approach for identifying candidate DNA adducts utilized the characteristic neutral loss of deoxyribose. In Paper I, the data generated from high resolution MS in Full Scan-Data independent Acquisition mode were analyzed manually using the fragmentation pattern of nucleoside adduct ions. In Paper II, processing time and efficiency were significantly improved by the development of nLossFinder software. Further, TraceFinder software was combined and used to evaluate and quantify detected adducts (Paper III). Another important development was a simultaneous approach for analysis of both DNA and RNA adducts in a single MS run presented in Paper IV. The analysis of amphipod samples based on the existing approaches and methods developed in this thesis showed that reproductive pathologies identified by the microscopic analysis were associated with specific DNA modifications that can be used to classify field-collected individuals according to their health status (Paper I). Moreover, epigenetic marks were the most influential adducts for this classification. Finally, the adduct profile of amphipods differed between the areas with r

Chemical reaction networks can be utilised as basic components for nucleic acid feedback control systems’ design for Synthetic Biology application. DNA hybridisation and programmed strand-displacement reactions are effective primitives for implementation. However, the experimental validation and scale-up of nucleic acid control systems are still considerably falling behind their theoretical designs. To aid with the progress heading into experimental implementations, we provide here chemical reaction networks that represent two fundamental classes of linear controllers: integral and static negative state feedback. We reduced the complexity of the networks by finding designs with fewer reactions and chemical species, to take account of the limits of current experimental capabilities and mitigate issues pertaining to crosstalk and leakage, along with toehold sequence design. The supplied control circuits are quintessential candidates for the first experimental validations of nucleic acid controllers, since they have a number of parameters, species, and reactions small enough for viable experimentation with current technical capabilities, but still represent challenging feedback control systems. They are also well suited to further theoretical analysis to verify results on the stability, performance, and robustness of this important new class of control systems. Keywords: feedback control; chemical reaction networks; nucleic acids; strand displacement circuits; synthetic biology

In this work, the synergistic/antagonistic impact of glucose and mixed starter cultures, namely Latilactobacillus sakei, Pediococcus pentosaceus, and Staphylococcus xylosus, was evaluated in Italian salami in terms of metab-olomics and sensory profiles. As expected, Salami manufactured with 0.5 % glucose exhibited a substantial pH drop, showing values close to 5 at 12 days of ripening. Metabolomics revealed 1841 metabolites, mainly belonging to amino acids, peptides, glycerolipids, and nucleic acids, showing a greater hierarchical role of glucose addition when compared with inoculated microbial starters. Distinct metabolomic fingerprints could be observed across treatments, mainly concerning glutamyl peptides like gamma-glutamyl-glutamate (related to the kokumi taste), biogenic amines (spermine), and lipid oxidation products (i.e., the oxylipin 13S-hydroperoxylino-lenic acid). Such differences may drive the differences in sensory profiles recorded among treatments. These findings indicate the need to select ad-hoc starter cultures to improve the safety, quality, and sensory traits of salami.

PAR1b is a cytoplasmic serine/threonine kinase that controls cell polarity and cell-cell interaction by regulating microtubule stability while mediating cytoplasmic-to-nuclear translocation of BRCA1. PAR1b is also a cellular target of the CagA protein of Helicobacter pylori, which leads to chronic infection causatively associated with the development of gastric cancer. The CagA-PAR1b interaction inactivates the kinase activity of PAR1b and thereby dampens PAR1b-mediated BRCA1 phosphorylation, which reduces the level of nuclear BRCA1 and thereby leads to BRCAness and BRCAness-associated genome instability underlying gastric carcinogenesis. While PAR1b can multimerize within the cells, little is known about the mechanism and functional role of PAR1b multimerization. We found in the present study that PAR1b was multimerized in vitro by binding with nucleic acids (both single- and double-stranded DNA/RNA) via the spacer region in a manner independent of nucleic-acid sequences, which markedly potentiated the kinase activity of PAR1b. Consistent with these in vitro observations, cytoplasmic introduction of double-stranded DNA or expression of single-stranded RNA increased the PAR1b kinase activity in the cells. These findings indicate that the cytoplasmic DNA/RNA contribute to nuclear accumulation of BRCA1 by constitutively activating/potentiating cytoplasmic PAR1b kinase activity, which is subverted in gastric epithelial cells upon delivery of H. pylori CagA oncoprotein.

PAR1b is a cytoplasmic serine/threonine kinase that controls cell polarity and cell-cell interaction by regulating microtubule stability while mediating cytoplasmic-to-nuclear translocation of BRCA1. PAR1b is also a cellular target of the CagA protein of Helicobacter pylori, which leads to chronic infection causatively associated with the development of gastric cancer. The CagA-PAR1b interaction inactivates the kinase activity of PAR1b and thereby dampens PAR1b-mediated BRCA1 phosphorylation, which reduces the level of nuclear BRCA1 and thereby leads to BRCAness and BRCAness-associated genome instability underlying gastric carcinogenesis. While PAR1b can multimerize within the cells, little is known about the mechanism and functional role of PAR1b multimerization. We found in the present study that PAR1b was multimerized in vitro by binding with nucleic acids (both single- and double-stranded DNA/RNA) via the spacer region in a manner independent of nucleic-acid sequences, which markedly potentiated the kinase activity of PAR1b. Consistent with these in vitro observations, cytoplasmic introduction of double-stranded DNA or expression of single-stranded RNA increased the PAR1b kinase activity in the cells. These findings indicate that the cytoplasmic DNA/RNA contribute to nuclear accumulation of BRCA1 by constitutively activating/potentiating cytoplasmic PAR1b kinase activity, which is subverted in gastric epithelial cells upon delivery of H. pylori CagA oncoprotein.

Targeting specific cells for sequencing is important for applications in cancer, microbiology, and infectious disease. Nucleic acid cytometry (NAC) is a powerful approach for accomplishing this because it allows specific cells to be isolated based on sequence biomarkers that are otherwise impossible to detect. However, existing methods require specialized microfluidic devices, limiting adoption. Here, a modified workflow is described that uses particle-templated emulsification (PTE) and flow cytometry to conduct the essential steps of cell detection and sorting normally accomplished by microfluidics. Our microfluidic-free workflow allows facile isolation and sequencing of cells, viruses, and nucleic acids and thus provides a powerful enrichment approach for targeted sequencing applications.

GeomBD3 is a robust Brownian dynamics simulation package designed to easily handle natural or engineered systems in diverse environments and arrangements. The software package described herein allows users to design, execute, and analyze BD simulations. The simulations use all-atom, rigid molecular models that diffuse according to overdamped Langevin dynamics and interact through electrostatic, Lennard-Jones, and ligand desolvation potentials. The program automatically calculates molecular association rates, surface residence times, and association statistics for any number of user-defined association criteria. Users can also extract molecular association pathways, diffusion coefficients, intermolecular interaction energies, intermolecular contact probability maps, and more using the provided supplementary analysis scripts. We detail the use of the package from start to finish and apply it to a protein-ligand system and a large nucleic acid biosensor. GeomBD3 provides a versatile tool for researchers from various disciplines that can aid in rational design of engineered systems or play an explanatory role as a complement to experiments. GeomBD version 3 is available on our website at http://chemcha-gpu0.ucr.edu/geombd3/ and KBbox at https://kbbox.h-its.org/toolbox/methods/molecular-simulation/geombd/.

The realization of electrochemical nucleic acid amplification tests (NAATs) at the point of care (POC) is highly desirable, but it remains a challenge given their high cost and lack of true portability/miniaturization. Here we show that mass-produced, industrial standardized, printed circuit boards (PCBs) can be repurposed to act as near-zero cost electrodes for self-assembled monolayer-based DNA biosensing, and further integration with a custom-designed and low-cost portable potentiostat. To show the analytical capability of this system, we developed a NAAT using isothermal recombinase polymerase amplification, bypassing the need of thermal cyclers, followed by an electrochemical readout relying on a sandwich hybridization assay. We used our sensor and device for analytical detection of the toxic microalgae Ostreopsis cf. ovata as a proof of concept. This work shows the potential of PCBs and open-source electronics to be used as powerful POC DNA biosensors at a low-cost., QC 20230523

Detection of small molecules is increasingly needed in environmental monitoring, food safety, medical research, among other industries. Allosteric transcription factors (aTFs) and aptamers have emerged as potential biorecognition elements to expand the biosensing toolkit and overcome limitations of rapid antibody-based detection. Rational design of signal transduction and amplification methods, and better understanding of bioreceptor-analyte interactions are paramount to build simple, efficient, affordable and reliable biosensing platforms. This dissertation showcases several unexplored and unaddressed factors that aid the development of rationally designed aTF- and aptamer-based biosensors. First, we introduce a novel endonuclease-mediated strand displacement circuit that is the first report of small molecule-aTFs interaction to actuate DNA toehold-mediated strand displacement reactions, which had been typically triggered by nucleic acid inputs only. This simple yet efficient mechanism was used to build tunable and modular biosensors for detecting nanomolar concentration of antibiotics in water samples in 20 minutes. Second, we introduce a computational pipeline that addresses fundamental issues in predicting aptamer-ligand interactions using modelling tools. This approach shows that using metastable representative structures extracted from multiple independent simulations can more accurately predict aptamer-small molecule interactions compared to conventional in silico approaches that assume that the bound and unbound aptamer conformations are the same. Third, we show how these predictions can support the design of aptasensors using an aflatoxin aptamer as an example. Through non-equilibrium competitive binding assays, we show that experimental results are in agreement with the hypothesized conformational selection binding pathway drawn from the computational simulations. Finally, we demonstrate the modularity of the signal amplifier previously developed by incorp

Nucleic acids are used as the template for protein synthesis, and this nature allows a wide range of biomedical applications. Developing new tools for efficient delivery of the exogenous nucleic acids into cells would expand the range of possible targets beyond what is generally accessible by conventional pharmaceutics. However, the clinical promise of nucleic acid is hindered by the poor cytosol delivery. For mRNA, the intact and functional mRNA is expected to be delivered into cytosol; for DNA, successful cytosol delivery is a pre-requisite for efficient transfection. Delivery difficulties drive the design of various delivering vehicles, categorized as viral and non-viral. The non-viral vectors appear to be the next generation tools for efficient delivery, but there is still much room for improvement in transfection efficiency compared to viruses. Therefore, it has been considered to take advantage of both systems by incorporating some features of the virus into non-viral delivery systems. Our lab has previously designed a platform to mimic viral capsid based on oligopeptide. Compared with the dominant lipid- and polymer-based systems, the peptide-based systems enable automated synthesis, allow singlestep formulation with nucleic acid, and have good biocompatibility. However, limited endosomal escape and poor cytosol delivery were the bottle neck for peptide-based systems, as in other non-viral systems. This thesis explored multiple ways for intracellular delivery in peptide-based systems, especially overcoming the endosomal escape bottle neck. Different methods have been incorporated, including peptide sequence modification, co-assembling procedure optimization, and surface modification of the assemblies. By understanding of the inter- and intra-molecular interactions within the assemblies’ structure, we discovered the significant alteration resulted by the subtle changes within the peptide sequence. In particular, the side-chain length and the neighboring atom (

DNA, the well-known molecule that carries the genetic information of almost all forms of life, represents a pivotal element in formulating intelligent and versatile micro/nanorobotic systems. DNA-functionalized micro/nanorobots have opened new and exciting opportunities in many research areas due to the synergistic combination of self-propulsion at the micro/nanoscale and the high specificity and programmability of DNA interactions. Here, their designs and applications are critically reviewed, which span from the use of DNA as the fuel to chemotactically power nanorobots toward cancer cells to DNA as the main building block for sophisticated phototactic biorobots, DNA nanodevices to self-monitor microrobots' activity status, DNA and RNA sensing, nucleic acids isolation, gene therapy, and water purification. The perspective on future directions of the field is also shared, envisioning DNA-mediated reconfigurable assemblies of nanorobotic swarms.

DNA, the well-known molecule that carries the genetic information of almost all forms of life, represents a pivotal element in formulating intelligent and versatile micro/nanorobotic systems. DNA-functionalized micro/nanorobots have opened new and exciting opportunities in many research areas due to the synergistic combination of self-propulsion at the micro/nanoscale and the high specificity and programmability of DNA interactions. Here, their designs and applications are critically reviewed, which span from the use of DNA as the fuel to chemotactically power nanorobots toward cancer cells to DNA as the main building block for sophisticated phototactic biorobots, DNA nanodevices to self-monitor microrobots' activity status, DNA and RNA sensing, nucleic acids isolation, gene therapy, and water purification. The perspective on future directions of the field is also shared, envisioning DNA-mediated reconfigurable assemblies of nanorobotic swarms.

PAR1b is a cytoplasmic serine/threonine kinase that controls cell polarity and cell-cell interaction by regulating microtubule stability while mediating cytoplasmic-to-nuclear translocation of BRCA1. PAR1b is also a cellular target of the CagA protein of Helicobacter pylori, which leads to chronic infection causatively associated with the development of gastric cancer. The CagA-PAR1b interaction inactivates the kinase activity of PAR1b and thereby dampens PAR1b-mediated BRCA1 phosphorylation, which reduces the level of nuclear BRCA1 and thereby leads to BRCAness and BRCAness-associated genome instability underlying gastric carcinogenesis. While PAR1b can multimerize within the cells, little is known about the mechanism and functional role of PAR1b multimerization. We found in the present study that PAR1b was multimerized in vitro by binding with nucleic acids (both single- and double-stranded DNA/RNA) via the spacer region in a manner independent of nucleic-acid sequences, which markedly potentiated the kinase activity of PAR1b. Consistent with these in vitro observations, cytoplasmic introduction of double-stranded DNA or expression of single-stranded RNA increased the PAR1b kinase activity in the cells. These findings indicate that the cytoplasmic DNA/RNA contribute to nuclear accumulation of BRCA1 by constitutively activating/potentiating cytoplasmic PAR1b kinase activity, which is subverted in gastric epithelial cells upon delivery of H. pylori CagA oncoprotein.

PAR1b is a cytoplasmic serine/threonine kinase that controls cell polarity and cell-cell interaction by regulating microtubule stability while mediating cytoplasmic-to-nuclear translocation of BRCA1. PAR1b is also a cellular target of the CagA protein of Helicobacter pylori, which leads to chronic infection causatively associated with the development of gastric cancer. The CagA-PAR1b interaction inactivates the kinase activity of PAR1b and thereby dampens PAR1b-mediated BRCA1 phosphorylation, which reduces the level of nuclear BRCA1 and thereby leads to BRCAness and BRCAness-associated genome instability underlying gastric carcinogenesis. While PAR1b can multimerize within the cells, little is known about the mechanism and functional role of PAR1b multimerization. We found in the present study that PAR1b was multimerized in vitro by binding with nucleic acids (both single- and double-stranded DNA/RNA) via the spacer region in a manner independent of nucleic-acid sequences, which markedly potentiated the kinase activity of PAR1b. Consistent with these in vitro observations, cytoplasmic introduction of double-stranded DNA or expression of single-stranded RNA increased the PAR1b kinase activity in the cells. These findings indicate that the cytoplasmic DNA/RNA contribute to nuclear accumulation of BRCA1 by constitutively activating/potentiating cytoplasmic PAR1b kinase activity, which is subverted in gastric epithelial cells upon delivery of H. pylori CagA oncoprotein.

Xeno-nucleic acids (XNAs) are synthetic genetic polymers with backbone structures composed of non-ribose or non-deoxyribose sugars. Phosphonomethylthreosyl nucleic acid (pTNA), a type of XNA that does not base pair with DNA or RNA, has been suggested as a possible genetic material for storing synthetic biology information in cells. A critical step in this process is the synthesis of XNA episomes using laboratory-evolved polymerases to copy DNA information into XNA. Here, we investigate the polymerase recognition of pTNA nucleotides using X-ray crystallography to capture the post-catalytic complex of engineered polymerases following the sequential addition of two pTNA nucleotides onto the 3'-end of a DNA primer. High-resolution crystal structures reveal that the polymerase mediates Watson-Crick base pairing between the extended pTNA adducts and the DNA template. Comparative analysis studies demonstrate that the sugar conformation and backbone position of pTNA are structurally more similar to threose nucleic acid than DNA even though pTNA and DNA share the same six-atom backbone repeat length. Collectively, these findings provide new insight into the structural determinants that guide the enzymatic synthesis of an orthogonal genetic polymer, and may lead to the discovery of new variants that function with enhanced activity.

Idiopathic chronic diarrhea (ICD) is a little understood common clinical problem in captive rhesus macaques claiming 33% of medical culls unrelated to research. The eukaryotic virome in digestive tract tissues collected at necropsy from nine animals with ICD was characterized using viral metagenomics. We compared the distribution of viral reads in tissues and mucosal scrapings from the stomach, duodenum, jejunum, ileum, and the proximal, transverse, and distal colons. In situ hybridization (ISH) using viral probes were performed on fixed tissues. Deep sequencing revealed multiple viruses in the Parvoviridae and Picornaviridae family. Tissues and mucosal scraping from the same locations showed closely related viral reads contents while different gut tissues from the same animal varied widely. ISH showed punctuated staining for both RNA and DNA viruses in the distal colon. Parvovirus staining was also detected in the stomach/duodenum/jejunum in distinct oval-shaped structures. The location of enteric viral nucleic acid differed widely between different viral families and along the length of the digestive tract.

Short segments of RNA displace one strand of a DNA duplex during diverse processes including transcription and CRISPR-mediated immunity and genome editing. These strand exchange events involve the intersection of two geometrically distinct helix types-an RNA:DNA hybrid (A-form) and a DNA:DNA homoduplex (B-form). Although previous evidence suggests that these two helices can stack on each other, it is unknown what local geometric adjustments could enable A-on-B stacking. Here we report the X-ray crystal structure of an RNA-5'/DNA-3' strand exchange junction at an anisotropic resolution of 1.6 to 2.2 Å. The structure reveals that the A-to-B helical transition involves a combination of helical axis misalignment, helical axis tilting and compression of the DNA strand within the RNA:DNA helix, where nucleotides exhibit a mixture of A- and B-form geometry. These structural principles explain previous observations of conformational stability in RNA/DNA exchange junctions, enabling a nucleic acid architecture that is repeatedly populated during biological strand exchange events.

Many of the ligands for Toll-like receptors (TLRs) are unique to microorganisms, such that receptor activation unequivocally indicates the presence of something foreign. However, a subset of TLRs recognizes nucleic acids, which are present in both the host and foreign microorganisms. This specificity enables broad recognition by virtue of the ubiquity of nucleic acids but also introduces the possibility of self-recognition and autoinflammatory or autoimmune disease. Defining the regulatory mechanisms required to ensure proper discrimination between foreign and self-nucleic acids by TLRs is an area of intense research. Progress over the past decade has revealed a complex array of regulatory mechanisms that ensure maintenance of this delicate balance. These regulatory mechanisms can be divided into a conceptual framework with four categories: compartmentalization, ligand availability, receptor expression and signal transduction. In this Review, we discuss our current understanding of each of these layers of regulation.

Nature evolves molecular interaction networks through persistent perturbation and selection, in stark contrast to drug discovery, which evaluates candidates one at a time by screening. Here, nature's highly parallel ligand-target search paradigm is recapitulated in a screen of a DNA-encoded library (DEL; 73,728 ligands) against a library of RNA structures (4,096 targets). In total, the screen evaluated ∼300 million interactions and identified numerous bona fide ligand-RNA three-dimensional fold target pairs. One of the discovered ligands bound a 5'GAG/3'CCC internal loop that is present in primary microRNA-27a (pri-miR-27a), the oncogenic precursor of microRNA-27a. The DEL-derived pri-miR-27a ligand was cell active, potently and selectively inhibiting pri-miR-27a processing to reprogram gene expression and halt an otherwise invasive phenotype in triple-negative breast cancer cells. By exploiting evolutionary principles at the earliest stages of drug discovery, it is possible to identify high-affinity and selective target-ligand interactions and predict engagements in cells that short circuit disease pathways in preclinical disease models.

Across biological scales, gene-regulatory networks employ autorepression (negative feedback) to maintain homeostasis and minimize failure from aberrant expression. Here, we present a proof of concept that disrupting transcriptional negative feedback dysregulates viral gene expression to therapeutically inhibit replication and confers a high evolutionary barrier to resistance. We find that nucleic-acid decoys mimicking cis-regulatory sites act as "feedback disruptors," break homeostasis, and increase viral transcription factors to cytotoxic levels (termed "open-loop lethality"). Feedback disruptors against herpesviruses reduced viral replication >2-logs without activating innate immunity, showed sub-nM IC50, synergized with standard-of-care antivirals, and inhibited virus replication in mice. In contrast to approved antivirals where resistance rapidly emerged, no feedback-disruptor escape mutants evolved in long-term cultures. For SARS-CoV-2, disruption of a putative feedback circuit also generated open-loop lethality, reducing viral titers by >1-log. These results demonstrate that generating open-loop lethality, via negative-feedback disruption, may yield a class of antimicrobials with a high genetic barrier to resistance.

ObjectiveWe sought to determine if corticosteroid administration is associated with a SARS-CoV-2 nucleic acid test-positive result and to describe therapies administered to SARS-CoV-2 infected children.MethodsWe collected cross-sectional data from participants recruited in 41 pediatric emergency departments (ED) in 10 countries between March 2020 and June 2021. Participants were <18 years old, had signs or symptoms of, or risk factors for acute SARS-CoV-2 infection, and had nucleic acid testing performed. To determine if SARS-CoV-2 test status was independently associated with corticosteroid administration, we used a multivariable conditional logistic regression model matched by study site to compare treatments administered based on SARS-CoV-2 test and disposition status. This analysis was repeated for the subgroup of study participants who were hospitalized.Results30.3% (3,121/10,315) of participants were SARS-CoV-2-positive. Although remdesivir was more commonly administered to SARS-CoV-2-positive children, use was infrequent (25/3120 [0.8%] vs 1/7188 [0.01%]; P = .001). Corticosteroid use was less common among SARS-CoV-2-positive children (219/3120 [7.0%] vs 759/7190 [10.6%]; P < .001). Among hospitalized children, there were no differences in provision of inotropes, respiratory support, chest drainage or extracorporeal membrane oxygenation between groups. Corticosteroid administration was associated with age, history of asthma, wheezing, study month, hospitalization and intensive care unit admission; it was not associated with a positive SARS-CoV-2 test result overall (aOR: 0.91; 95%CI: 0.74, 1.12) or among the subgroup of those hospitalized (aOR: 1.04; 95%CI: 0.75, 1.44).ConclusionsFew disease-specific treatments are provided to SARS-CoV-2-positive children; clinical trials evaluating therapies in children are urgently needed.

Nature evolves molecular interaction networks through persistent perturbation and selection, in stark contrast to drug discovery, which evaluates candidates one at a time by screening. Here, nature's highly parallel ligand-target search paradigm is recapitulated in a screen of a DNA-encoded library (DEL; 73,728 ligands) against a library of RNA structures (4,096 targets). In total, the screen evaluated ∼300 million interactions and identified numerous bona fide ligand-RNA three-dimensional fold target pairs. One of the discovered ligands bound a 5'GAG/3'CCC internal loop that is present in primary microRNA-27a (pri-miR-27a), the oncogenic precursor of microRNA-27a. The DEL-derived pri-miR-27a ligand was cell active, potently and selectively inhibiting pri-miR-27a processing to reprogram gene expression and halt an otherwise invasive phenotype in triple-negative breast cancer cells. By exploiting evolutionary principles at the earliest stages of drug discovery, it is possible to identify high-affinity and selective target-ligand interactions and predict engagements in cells that short circuit disease pathways in preclinical disease models.

Nucleic acids have become viable prognostic and diagnostic biomarkers for a diverse class of diseases, particularly cancer. However, the low femtomolar to attomolar concentration of nucleic acids in human samples require sensors with excellent detection capabilities; many past and current platforms fall short or are economically difficult. Strand-mediated signal amplifiers such as hybridization chain reaction (HCR) and catalytic hairpin assembly (CHA) are superior methods for detecting trace amounts of biomolecules because one target molecule triggers the continuous production of synthetic double-helical DNA. This cascade event is highly discriminatory to the target via sequence specificity, and it can be coupled with fluorescence, electrochemistry, magnetic moment, and electrochemiluminescence for signal reporting. Here, we review recent advances in enhancing the sensing abilities in HCR and CHA for improved live-cell imaging efficiency, lowered limit of detection, and optimized multiplexity. We further outline the potential for clinical translatability of HCR and CHA by summarizing progress in employing these two tools for in vivo imaging, human sample testing, and sensing-treating dualities. We finally discuss their future prospects and suggest clinically-relevant experiments to supplement further related research.

Although mammalian embryo development depends on critical protein isoforms that arise from embryo-specific nucleic acid modifications, the role of these isoforms is not yet clear. Challenges arise in measuring protein isoforms and nucleic acids from the same single embryos and blastomeres. Here we present a multimodal technique for performing same-embryo nucleic acid and protein isoform profiling (single-embryo nucleic acid and protein profiling immunoblot, or snapBlot). The method integrates protein isoform measurement by fractionation polyacrylamide gel electrophoresis (fPAGE) with off-chip analysis of nucleic acids from the nuclei. Once embryos are harvested and cultured to the desired stage, they are sampled into the snapBlot device and subjected to fPAGE. After fPAGE, 'gel pallets' containing nuclei are excised from the snapBlot device for off-chip nucleic acid analyses. fPAGE and nuclei analyses are indexed to each starting sample, yielding same-embryo multimodal measurements. The entire protocol, including processing of samples and data analysis, takes 2-3 d. snapBlot is designed to help reveal the mechanisms by which embryo-specific nucleic acid modifications to both genomic DNA and messenger RNA orchestrate the growth and development of mammalian embryos.

We present a Chemistry and Structure Screen Integrated Efficiently (CASSIE) approach (named for Greek prophet Cassandra) to design inhibitors for cancer biology and pathogenesis. CASSIE provides an effective path to target master keys to control the repair-replication interface for cancer cells and SARS CoV-2 pathogenesis as exemplified here by specific targeting of Poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribose glycohydrolase ARH3 macrodomains plus SARS CoV-2 nonstructural protein 3 (Nsp3) Macrodomain 1 (Mac1) and Nsp15 nuclease. As opposed to the classical massive effort employing libraries with large numbers of compounds against single proteins, we make inhibitor design for multiple targets efficient. Our compact, chemically diverse, 5000 compound Goldilocks (GL) library has an intermediate number of compounds sized between fragments and drugs with predicted favorable ADME (absorption, distribution, metabolism, and excretion) and toxicological profiles. Amalgamating our core GL library with an approved drug (AD) library, we employ a combined GLAD library virtual screen, enabling an effective and efficient design cycle of ranked computer docking, top hit biophysical and cell validations, and defined bound structures using human proteins or their avatars. As new drug design is increasingly pathway directed as well as molecular and mechanism based, our CASSIE approach facilitates testing multiple related targets by efficiently turning a set of interacting drug discovery problems into a tractable medicinal chemistry engineering problem of optimizing affinity and ADME properties based upon early co-crystal structures. Optimization efforts are made efficient by a computationally-focused iterative chemistry and structure screen. Thus, we herein describe and apply CASSIE to define prototypic, specific inhibitors for PARG vs distinct inhibitors for the related macrodomains of ARH3 and SARS CoV-2 Nsp3 plus the SARS CoV-2 Nsp15 RNA nuclease.

Glioblastoma (GBM) is one of the most difficult cancers to effectively treat, in part because of the lack of precision therapies and limited therapeutic access to intracranial tumor sites due to the presence of the blood-brain and blood-tumor barriers. We have developed a precision medicine approach for GBM treatment that involves the use of brain-penetrant RNA interference-based spherical nucleic acids (SNAs), which consist of gold nanoparticle cores covalently conjugated with radially oriented and densely packed small interfering RNA (siRNA) oligonucleotides. On the basis of previous preclinical evaluation, we conducted toxicology and toxicokinetic studies in nonhuman primates and a single-arm, open-label phase 0 first-in-human trial (NCT03020017) to determine safety, pharmacokinetics, intratumoral accumulation and gene-suppressive activity of systemically administered SNAs carrying siRNA specific for the GBM oncogene Bcl2Like12 (Bcl2L12). Patients with recurrent GBM were treated with intravenous administration of siBcl2L12-SNAs (drug moniker: NU-0129), at a dose corresponding to 1/50th of the no-observed-adverse-event level, followed by tumor resection. Safety assessment revealed no grade 4 or 5 treatment-related toxicities. Inductively coupled plasma mass spectrometry, x-ray fluorescence microscopy, and silver staining of resected GBM tissue demonstrated that intravenously administered SNAs reached patient tumors, with gold enrichment observed in the tumor-associated endothelium, macrophages, and tumor cells. NU-0129 uptake into glioma cells correlated with a reduction in tumor-associated Bcl2L12 protein expression, as indicated by comparison of matched primary tumor and NU-0129-treated recurrent tumor. Our results establish SNA nanoconjugates as a potential brain-penetrant precision medicine approach for the systemic treatment of GBM.

G-quadruplexes (GQs) are highly stable noncanonical nucleic acid structures that form in the DNA of human cells and play fundamental roles in maintaining genomic stability and regulating gene expression. These unique structures exert broad influence over biologically important processes and can modulate cell survival and human health. In fact, mutations, hyper-stability, and dissociation of GQs are implicated in neurodegenerative disease, mental retardation, premature-aging conditions, and various cancers. As such, GQs are novel drug targets. GQ-targeting therapeutics are developed to influence the folding and genetic interactions of GQs that are implicated in diseased states. To do so requires a greater understanding of GQ structure and dynamics and molecular dynamics (MD) simulations are well suited to provide these fundamental insights. Previous MD simulations of GQs have provided limited information due to inaccuracies in their models, namely the nonpolarizable nature of their force fields (FFs). The cutting-edge Drude polarizable FF models electronic degrees of freedom, allowing charge distribution to change in response to its environment. This is an important component for modeling ion-ion and ion-DNA interactions and can influence the overall stability of GQ structures. The work herein employs the Drude polarizable FF to 1) describe the role of electronic structure on the dynamics and folded stability of GQs, 2) determine the impact of ion interaction on GQ stability, and 3) characterize the role of G-hairpin motifs in GQ intermediates. Such fundamental investigations will help clarify GQs role in healthy and diseased states and transform our understanding of noncanonical DNA, improving human health, therapeutic design, and fundamental science.

The formation of calixarene-based liposomes was investigated, and the characterization of these nanostructures was carried out using several techniques. Four amphiphilic calixarenes were used. The length of the hydrophobic chains attached to the lower rim as well as the nature of the polar group present in the upper rim of the calixarenes were varied. The lipid bilayer was formed with one calixarene and with the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE. The cytotoxicity of the liposomes for various cell lines was also studied. From the results obtained, the liposomes formed with the least cytotoxic calixarene, (TEAC12)4, were used as nanocarriers of both nucleic acids and the antineoplastic drug doxorubicin, DOX. Results showed that (TEAC12)4/DOPE/p-EGFP-C1 lipoplexes, of a given composition, can transfect the genetic material, although the transfection efficiency substantially increases in the presence of an additional amount of DOPE as coadjuvant. On the other hand, the (TEAC12)4/DOPE liposomes present a high doxorubicin encapsulation efficiency, and a slow controlled release, which could diminish the side effects of the drug.

Targeted sequencing enables sensitive and cost-effective analysis by focusing resources on molecules of interest. Existing methods, however, are limited in enrichment power and target capture length. Here, we present a novel method that uses compound nucleic acid cytometry to achieve million-fold enrichments of molecules >10 kbp in length using minimal prior target information. We demonstrate the approach by sequencing HIV proviruses in infected individuals. Our method is useful for rare target sequencing in research and clinical applications, including for identifying cancer-associated mutations or sequencing viruses infecting cells.

Current health emergencies have highlighted the need to have rapid, sensitive, and convenient platforms for the detection of specific antibodies. In response, we report here the design of an electrochemical DNA circuit that responds quantitatively to multiple specific antibodies. The approach employs synthetic antigen-conjugated nucleic acid strands that are rationally designed to induce a strand displacement reaction and release a redox reporter-modified strand upon the recognition of a specific target antibody. The approach is sensitive (low nanomolar detection limit), specific (no signal is observed in the presence of non-targeted antibodies), and selective (the platform can be employed in complex media, including 90% serum). The programmable nature of the strand displacement circuit makes it also versatile, and we demonstrate here the detection of five different antibodies, including three of which are clinically relevant. Using different redox reporters, we also show that the antibody-responsive circuit can be multiplexed and responds to different antibodies in the same solution without crosstalk.

The formation of calixarene-based liposomes was investigated, and the characterization of these nanostructures was carried out using several techniques. Four amphiphilic calixarenes were used. The length of the hydrophobic chains attached to the lower rim as well as the nature of the polar group present in the upper rim of the calixarenes were varied. The lipid bilayer was formed with one calixarene and with the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE. The cytotoxicity of the liposomes for various cell lines was also studied. From the results obtained, the liposomes formed with the least cytotoxic calixarene, (TEAC12)4, were used as nanocarriers of both nucleic acids and the antineoplastic drug doxorubicin, DOX. Results showed that (TEAC12)4/DOPE/p-EGFP-C1 lipoplexes, of a given composition, can transfect the genetic material, although the transfection efficiency substantially increases in the presence of an additional amount of DOPE as coadjuvant. On the other hand, the (TEAC12)4/DOPE liposomes present a high doxorubicin encapsulation efficiency, and a slow controlled release, which could diminish the side effects of the drug.

The ability to detect and quantify viral nucleic acids is of paramount significance for diagnostics, therapeutics, and vaccine development. To combat globally spreading infectious diseases such as SARS-CoV-2, Ebola, and Zika, rapid and sensitive nucleic acid detection and quantification methods are in grave need for development. Compared with conventional tube-based assays, the digital format of bulk assays (e.g., digital PCR or digital LAMP) holds the potential of enabling absolute quantification of nucleic acids with no dependency on endogenous references or standards. Unlike hot-start digital PCR and LAMP, the implementation of digital quantification of the near-ambient-temperature assays using RPA and CRISPR may suffer from premature target amplification. Droplet microfluidics offers versatile yet controllable handling of samples and reagents, which provides confined compartments for enhanced assays kinetics and can be exploited to effectively circumvent premature amplification before droplet partition. In this thesis, we aim to develop effective yet facile microfluidic technology for reagent handling and manipulation with droplets. Firstly, we developed a Picoinjection Aided Digital reaction unLOCKing (PADLOCK) assay for ultrasensitive nucleic acid quantification. PADLOCK is realized through partitioning MgOAc-deprived RPA assays into picolitre water-in-oil droplets, the digital reaction unlocking is assisted by a downstream microfluidic injector loaded with the reaction initiator MgOAc. Hence, premature amplification in bulk is handily circumvented. To provide an in-depth view, we investigated three sensing schemes including CRISPR-Cas13a sensing, exo probe sensing, and EvaGreen sensing with PADLOCK-CRISPR and PADLOCK-EXO achieving sensitivity at single molecule level. We further demonstrated the utility of PADLOCK-CRISPR assay for HPV16 viral loads quantification in clinical samples, which generated 100% concordant results with qPCR. We believe the thorough i

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), is a prokaryotic adaptive immune system against viruses and plasmids from most of the archaea and bacteria. Recently, isothermal assays with CRISPR, such as Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), have been proved to enable rapid and efficient nucleic acid detection which is promising for the development of the point-of-care testing systems. The elegant trans cleavage reporter unlocking mechanism built on the programmable crRNA guided CRISPR nucleases ensure superior specificity and sensitivity. However, the typical CRISPR-based approaches separate the amplification and detection into a two-step process which not only induces potential contamination but also increases the complexity using digital quantification. This thesis addresses the challenges in developing all-in-one CRISPR assays that are compatible with isothermal recombinase polymerase amplification and rolling circle amplification respectively, and implementation of the digital detection of these assays for absolute quantification in combination with microfluidic technology. In the first project, we first developed all-in-one SHERLOCK with enhanced molecular mobility so as to improve the reaction robustness. We further applied this one-pot SHERLOCK assay using droplet microfluidics for digital quantification. The Microfluidics-Enabled Digital Isothermal Cas13a Assay (MEDICA), a digital format of one-pot SHERLOCK, leverages microfluidic technology to allow the separation of the magnesium initiator from the target-included master mix, allowing MEDICA to completely eliminated undesired premature amplification, as well as the compartmentation of the assay into picoliter reactions for absolute quantification by direct counting of the droplet numbers. MEDICA demonstrated single-molecule detection ability, more rapid and stable qualitative (10 min) and quantitative (25 min) results. We also demonstrated that MEDICA succ

Nucleic acid detection is of great importance in a variety of areas, from life science and clinical diagnosis to environmental monitoring and food safety. Unfortunately, nucleic acid targets are always found in trace amounts and their response signals are difficult to be detected. Amplification mechanisms are then practically needed to either duplicate nucleic acid targets or enhance the detection signals. Polymerase chain reaction (PCR) is one of the most popular and powerful techniques for nucleic acid analysis, serving as a gold standard for detection. But the requirement of costly devices for precise thermo-cycling procedures in PCR has severely hampered the wide applications of PCR. Fortunately, isothermal molecular reactions have emerged as promising alternatives. The past decade has witnessed significant progress in the research of isothermal molecular reactions utilizing hairpin DNA probes (HDPs). Based on the nucleic acid strand interaction mechanisms, the hairpin DNA-mediated isothermal amplification (HDMIA) techniques can be mainly divided into three categories: strand assembly reactions, strand decomposition reactions, and strand creation reactions. This thesis introduces the HDMIA methods with ultra-high performances. Their sensing principles and advanced designs are evaluated, along with their wide applications, especially those benefiting from the utilization of G-quadruplexes and nanomaterials. The thesis also discusses the current challenges encountered, highlights the potential solutions, and points out the possible future directions. The first project in Chapter 2 addresses ultrasensitive fluorescent strategy for DNA detection. The method discussed utilizes a molecular beacon, hairpin DNA probe, and a nicking enzyme to trigger dual-cycling reactions, showing ultra-sensitivity, and very high selectivity over mismatched and random DNA sequences. Chapter 3 focuses attention on the utilization of advanced material to trigger dual-signal amplification

Targeted sequencing enables sensitive and cost-effective analysis by focusing resources on molecules of interest. Existing methods, however, are limited in enrichment power and target capture length. Here, we present a novel method that uses compound nucleic acid cytometry to achieve million-fold enrichments of molecules >10 kbp in length using minimal prior target information. We demonstrate the approach by sequencing HIV proviruses in infected individuals. Our method is useful for rare target sequencing in research and clinical applications, including for identifying cancer-associated mutations or sequencing viruses infecting cells.

The formation of calixarene-based liposomes was investigated, and the characterization of these nanostructures was carried out using several techniques. Four amphiphilic calixarenes were used. The length of the hydrophobic chains attached to the lower rim as well as the nature of the polar group present in the upper rim of the calixarenes were varied. The lipid bilayer was formed with one calixarene and with the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE. The cytotoxicity of the liposomes for various cell lines was also studied. From the results obtained, the liposomes formed with the least cytotoxic calixarene, (TEAC12)4, were used as nanocarriers of both nucleic acids and the antineoplastic drug doxorubicin, DOX. Results showed that (TEAC12)4/DOPE/p-EGFP-C1 lipoplexes, of a given composition, can transfect the genetic material, although the transfection efficiency substantially increases in the presence of an additional amount of DOPE as coadjuvant. On the other hand, the (TEAC12)4/DOPE liposomes present a high doxorubicin encapsulation efficiency, and a slow controlled release, which could diminish the side effects of the drug.

The formation of calixarene-based liposomes was investigated, and the characterization of these nanostructures was carried out using several techniques. Four amphiphilic calixarenes were used. The length of the hydrophobic chains attached to the lower rim as well as the nature of the polar group present in the upper rim of the calixarenes were varied. The lipid bilayer was formed with one calixarene and with the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE. The cytotoxicity of the liposomes for various cell lines was also studied. From the results obtained, the liposomes formed with the least cytotoxic calixarene, (TEAC12)4, were used as nanocarriers of both nucleic acids and the antineoplastic drug doxorubicin, DOX. Results showed that (TEAC12)4/DOPE/p-EGFP-C1 lipoplexes, of a given composition, can transfect the genetic material, although the transfection efficiency substantially increases in the presence of an additional amount of DOPE as coadjuvant. On the other hand, the (TEAC12)4/DOPE liposomes present a high doxorubicin encapsulation efficiency, and a slow controlled release, which could diminish the side effects of the drug.

Nucleic acid detection is of great importance in a variety of areas, from life science and clinical diagnosis to environmental monitoring and food safety. Unfortunately, nucleic acid targets are always found in trace amounts and their response signals are difficult to be detected. Amplification mechanisms are then practically needed to either duplicate nucleic acid targets or enhance the detection signals. Polymerase chain reaction (PCR) is one of the most popular and powerful techniques for nucleic acid analysis, serving as a gold standard for detection. But the requirement of costly devices for precise thermo-cycling procedures in PCR has severely hampered the wide applications of PCR. Fortunately, isothermal molecular reactions have emerged as promising alternatives. The past decade has witnessed significant progress in the research of isothermal molecular reactions utilizing hairpin DNA probes (HDPs). Based on the nucleic acid strand interaction mechanisms, the hairpin DNA-mediated isothermal amplification (HDMIA) techniques can be mainly divided into three categories: strand assembly reactions, strand decomposition reactions, and strand creation reactions. This thesis introduces the HDMIA methods with ultra-high performances. Their sensing principles and advanced designs are evaluated, along with their wide applications, especially those benefiting from the utilization of G-quadruplexes and nanomaterials. The thesis also discusses the current challenges encountered, highlights the potential solutions, and points out the possible future directions. The first project in Chapter 2 addresses ultrasensitive fluorescent strategy for DNA detection. The method discussed utilizes a molecular beacon, hairpin DNA probe, and a nicking enzyme to trigger dual-cycling reactions, showing ultra-sensitivity, and very high selectivity over mismatched and random DNA sequences. Chapter 3 focuses attention on the utilization of advanced material to trigger dual-signal amplification