Beyond DNA origami: the unfolding prospects of nucleic acid nanotechnology

Nucleic acid nanotechnology exploits the programmable molecular recognition properties of natural and synthetic nucleic acids to assemble structures with nanometer‐scale precision. In 2006, DNA origami transformed the field by providing a versatile platform for self‐assembly of arbitrary shapes from one long DNA strand held in place by hundreds of short, site‐specific (spatially addressable) DNA ‘staples’. This revolutionary approach has led to the creation of a multitude of two‐dimensional and three‐dimensional scaffolds that form the basis for functional nanodevices. Not limited to nucleic acids, these nanodevices can incorporate other structural and functional materials, such as proteins and nanoparticles, making them broadly useful for current and future applications in emerging fields such as nanomedicine, nanoelectronics, and alternative energy. WIREs Nanomed Nanobiotechnol 2012, 4:139–152. doi: 10.1002/wnan.170 For further resources related to this article, please visit the WIREs website .Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90282/1/170_ftp.pd

Lab-on-a-Chip Fabrication and Application

The necessity of on-site, fast, sensitive, and cheap complex laboratory analysis, associated with the advances in the microfabrication technologies and the microfluidics, made it possible for the creation of the innovative device lab-on-a-chip (LOC), by which we would be able to scale a single or multiple laboratory processes down to a chip format. The present book is dedicated to the LOC devices from two points of view: LOC fabrication and LOC application

Doctor of Philosophy

dissertationPeptide nucleic acid (PNA) is a nucleic acid mimic that shows tremendous potential for use in therapeutic and biosensing applications due to its high binding affinity for DNA and RNA and its excellent biostability. The therapeutic potential of PNA is hindered, however, by poor cellular uptake, solubility, and bioavailability. Although various approaches have been taken to overcome these critical limitations and realize the full potential of PNA, more efficient solutions are still desired. We hypothesize that negatively charged PNA analogues would electrostatically mimic DNA and RNA, thus overcoming the limitations mentioned above. This dissertation is mainly focused on our initial studies to investigate the tolerance of the PNA structure to the addition of negatively charged side chains. We explored the effect of ionic strength on binding affinity for modified PNAs having either negatively charged side chains or positively charged side chains (Chapter 2). We observed that as ionic strength is increased, negatively charged PNA increases in affinity for DNA and RNA, whereas positively charged PNA decreases in affinity for DNA and RNA. The point at which these trends intersect hovers near physiological salt concentration. In a simulated physiological buffer, negatively charged PNA shows slightly higher affinity for RNA whereas positively charged PNA shows slightly higher affinity for DNA. Intrigued by the effect of side chain structure and electrostatics on binding affinity, we were also curious to explore the mismatch and orientation selectivity of these y-substituted PNAs (Chapter 3). We observed that positively charged side chains provide higher selectivity in DNA binding, while negatively charged side chains provide higher selectivity in RNA binding. Our results provide insight into the impact of side chain structure and electrostatics on the binding affinity and selectivity with DNA and RNA under physiological conditions. Since PNA can be negatively charged without sacrificing binding affinity and selectivity, we anticipate that these molecules will show promise as therapeutics that take advantage of both the inherent benefits of PNA and the multitude of charge-based delivery technologies currently being developed for DNA and RNA. PNA also shows promise for use in synthetic biology applications, but the evolution of abiotic polymers such as PNA requires methods for sequence encoding and amplification. Chapter 4 describes our efforts to synthesize a modified PNA monomer that is designed to polymerize using dynamic reaction conditions. DNA-based micelles have the potential to be used as stimuli-responsive materials due to their ability to undergo programmable assembly and disassembly. Chapter 5 outlines our synthesis of a potential multivalent micellar scaffold

Inspirations of biomimetic affinity ligands: a review

Affinity chromatography is a well-known method dependent on molecular recognition and is used to purify biomolecules by mimicking the specific interactions between the biomolecules and their substrates. Enzyme substrates, cofactors, antigens, and inhibitors are generally utilized as bioligands in affinity chromatography. However, their cost, instability, and leakage problems are the main drawbacks of these bioligands. Biomimetic affinity ligands can recognize their target molecules with high selectivity. Their cost-effectiveness and chemical and biological stabilities make these antibody analogs favorable candidates for affinity chromatography applications. Biomimetics applies to nature and aims to develop nanodevices, processes, and nanomaterials. Today, biomimetics provides a design approach to the biomimetic affinity ligands with the aid of computational methods, rational design, and other approaches to meet the requirements of the bioligands and improve the downstream process. This review highlighted the recent trends in designing biomimetic affinity ligands and summarized their binding interactions with the target molecules with computational approaches

A Brief History, Status, and Perspective of Modified Oligonucleotides for Chemotherapeutic Applications

The advent of rapid and efficient methods of oligonucleotide synthesis has allowed the design of modified oligonucleotides that are complementary to specific nucleotide sequences in mRNA targets. Such modified oligonucleotides can be used to disrupt the flow of genetic information from transcribed mRNAs to proteins. This antisense strategy has been used to develop therapeutic oligonucleotides against cancer and various infectious diseases in humans. This overview reports recent advances in the application of oligonucleotides as drug candidates, describes the relationship between oligonucleotide modifications and their therapeutic profiles, and provides general guidelines for enhancing oligonucleotide drug properties.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143788/1/cpnc0401.pd

A REVIEW ON APPLICATION OF NANOADJUVANT AS DELIVERY SYSTEM

Worldwide immunization can save millions of peoples to lives year by using the vaccines. The subunit of antigen components is manufactured which can stimulate the immune system by providing specific immunity against specific diseases. Subunit vaccines have many advantages like as high safety profile but having limited ability to provide immunogenicity. These traditional subunit vaccines activate only innate immunity, encourage cell-mediated transport of antigen to lymphoid tissues. Newly nano-adjuvants based vaccines carrier systems like liposomes, virosome, micelles, polymeric particles, protein, and peptides are developed by using various substances like viral proteins, polymer and polystyrene having immanent adjuvanticity and also provide exalted capability in manufacturing subunit vaccines. It has chromospheres substances that have various properties such as targeted, anti-damaging and caliber to lead immune reactions towards Th1 and Th2 route, which is an important feature for humoral as well as cellular immunity. The whole thing based on the carrier system, the role of nano-adjuvants, its pharmacokinetics and distribution in the body system. It has the ability to provide antigen-specific immunity to both systemic as well as mucosal by different vaccination passage. Also, the nano-adjuvants based vaccine suggested that direct targeting of antigen to improve the vaccine potency without sacrificing safety

Optimization and validation of a novel direct-lysis differential extraction procedure

Forensic analysis of DNA from sexual assault kits is a laborious process. These samples may be a mixture of sperm and male or female epithelial cells (E-cells). Generally, it is the sperm cells that are of greatest forensic value. Since its introduction in 1985 by Gill, Jefferys and Warrett, differential extraction has remained an essential pre-PCR extraction procedure adopted by most forensic laboratories for the preferential lysis of E-cells and isolation of sperm cells/male fraction prior to DNA profiling. The differential extraction procedure operates based on the packaging of DNA in these two types of cells. The E cells are first lysed by sodium dodecyl sulfate (SDS) and Proteinase K which leaves the sperm cells intact. The mixture is centrifuged leaving E-cell DNA in the supernatant and sperm cells in the pellet. After several wash steps to remove residual E cell DNA, the sperm fraction is then subjected to lysis using SDS, proteinase K, and dithiothreitol (DTT). DTT reduces the disulfide bonds present in the sperm nucleus, thereby releasing sperm cell DNA. The traditional Gill method of differential extraction, while proven to be highly effective in providing two separate fractions for a simplified interpretation of profiles, is a labor intensive and time-consuming process, requiring approximately six hours of an analyst’s concentration. In a casework scenario where an evidence sample is of a higher E cell concentration compared to sperm cells, it is inevitable to obtain mixture profiles that becomes more difficult to interpret. To mitigate carryover from the female fraction, the sperm cell fraction is usually subjected to multiple wash steps. Furthermore, the resulting fractions must be subjected to additional pre-PCR DNA purification procedures to remove PCR inhibitors such as SDS and Proteinase K which result in varying degrees on DNA loss. Progress has been made over the years to introduce methods that allow for PCR-ready lysates without additional purification steps, often referred to as direct lysis methods. However, none have been proven to be viable options for use in sexual assault samples. Our laboratory has developed a novel differential extraction procedure that is not only time-efficient and less laborious but also utilizes a direct-lysis procedure requiring no further pre-PCR purification for most samples. The novel procedure uses ZyGEM, which contains the thermophilic EA1 protease proven to effectively digest biological samples and produce PCR-ready lysates suitable for downstream nucleic acid amplification, thereby minimizing DNA loss. The procedure uses a multi-enzymatic approach and utilizes the different optimal activity temperatures of the enzymes to perform most of the process in a DNA extraction lab thermocycler, requiring only a single centrifugation for the usual separation of the E-cell fraction and no subsequent washing steps for the sperm cell fraction. It has the potential to be a rapid, robust procedure that can be easily implemented in any forensic laboratory. This thesis will describe the procedure and report progress in the procedure optimization.2019-10-24T00:00:00

Proto‐Urea‐RNA (Wöhler RNA) Containing Unusually Stable Urea Nucleosides

The RNA world hypothesis assumes that life on Earth began with nucleotides that formed information‐carrying RNA oligomers able to self‐replicate. Prebiotic reactions leading to the contemporary nucleosides are now known, but their execution often requires specific starting materials and lengthy reaction sequences. It was therefore proposed that the RNA world was likely proceeded by a proto‐RNA world constructed from molecules that were likely present on the early Earth in greater abundance. Herein, we show that the prebiotic starting molecules bis‐urea (biuret) and tris‐urea (triuret) are able to directly react with ribose. The urea‐ribosides are remarkably stable because they are held together by a network of intramolecular, bifurcated hydrogen bonds. This even allowed the synthesis of phosphoramidite building blocks and incorporation of the units into RNA. Investigations of the nucleotides’ base‐pairing potential showed that triuret:G RNA base pairs closely resemble U:G wobble base pairs. Based on the probable abundance of urea on the early Earth, we postulate that urea‐containing RNA bases are good candidates for a proto‐RNA world