1,720,996 research outputs found
Increased duplex stabilization in porphyrin-LNA zipper arrays with structure dependent exciton coupling
No full textData files containing the melting curves of modified DNA, and fitting procedures to extract thermodynamic data.
This dataset was used in:
Singleton DG, Hussain R, Siligardi G, Kumar P, Hrdlicka PJ, Berova N and Stulz E (2015) Increased duplex stabilization in porphyrin-LNA zipper arrays with structure dependent exciton coupling. Organic & Biomolecular Chemistry, DOI: 10.1039/C5OB01681A </span
Increased duplex stabilization in porphyrin-LNA zipper arrays with structure dependent exciton coupling
No full textPorphyrins were attached to LNA uridine building blocks via rigid 5-acetylene or more flexible propargyl-amide linkers and incorporated into DNA strands. The systems show a greatly increased thermodynamic stability when using as little as three porphyrins in a zipper arrangement. Thermodynamic analysis reveals clustering of the strands into more ordered duplexes with both greater negative ??S and ??H values, and less ordered duplexes with small positive ??S differences, depending on the combination of linkers used. The exciton coupling between the porphyrins is dependent on the flanking DNA sequence in the single stranded form, and on the nature of the linker between the nucleobase and the porphyrin in the double stranded form; it is, however, also strongly influenced by intermolecular interactions. This system is suitable for the formation of stable helical chromophore arrays with sequence and structure dependent exciton coupling
Synthesis and Characterization of Energetically Activated Duplexes for Sequence-Unrestricted Recognition of Double-stranded DNA
No full textThe main purpose of the work described in this dissertation is to develop oligonucleotide-based probes that can target genomic DNA. The development of probes capable of interrupting the flow of genetic information in living organisms have become an interesting field of research due to their potential as diagnostic and fundamental research tools, and - the grand challenge - therapeutics that can combat diseases of genetic origin. There is an extensive need to expand the current toolbox of double-stranded DNA (dsDNA) targeting probes to enable high specificity targeting at physiologically relevant conditions without sequence limitations. The Hrdlicka lab focuses on the development of a novel DNA targeting methodology utilizing energetically activated DNA duplexes, which potentially overcome the limitations of current DNA recognition strategies (e.g., triplex-forming oligonucleotides, polyamides, and peptide nucleic acids). This approach originally utilized N2'-pyrene-functionalized 2'-amino-alpha-L-LNA nucleotides as the key activating modifications. However, these building blocks are synthetically difficult to make impeding the full characterization of this novel DNA recognition strategy. Identification of simpler and more readily accessible scaffolds therefore presented itself as a highly desirable goal in order to conduct structure-property relationship studies with the aim of optimizing the dsDNA binding affinity of Invader probes. The work presented in this dissertation describes the synthesis and characterization of oligonucleotides and Invader probes based on (i) N2'-pyrene-functionalized 2'-amino-alpha-L-LNA adenosine, (ii) N2'-pyrene-/perylene-/coronene-functionalized 2'-N-methyl-2'-aminouridine monomers, to study the influence of intercalator size on dsDNA recognition efficiency, (iii) phosphorothioate DNA backbones, to improve pharmacokinetic properties, (iv) S2'-pyrene-functionalized 2'-thiouridine, to study the effect of electronegativity of the 2'-sugar atom on DNA recognition efficiency, (v) pseudo-complementary Invader building blocks, to further increase the binding affinity of Invader probes. The long-term goal of this research project is to develop simple nucleic acid probes that allow for sequence-unrestricted targeting of double-stranded DNA and to apply these probes as tools in molecular biology, nucleic acid diagnostics, and novel gene therapeutics.Thesis (Ph.D., Chemistry) -- University of Idaho, 201
Synthesis and Characterization of Nucleobase Functionalized Locked Nucleic Acids (LNAs); Chemical Modifications for therapeutic and diagnostic purposes.
No full textDNA is the genetic code of life, containing vital information required for the existence of life. An understanding of the structure of DNA, its function and Watson-Crick hybridization led to the conceptualization of the antisense strategy. The concept suggested that it is possible to design drugs that target disease-related mRNA (messenger RNA) antisense oligonucleotides (ASO) that bind to the target based on Watson-Crick hybridization rules and prevent its translation into proteins. Since, copy of target RNA present in a cell is much smaller in comparison to proteins, i.e., the most common drug target for small molecue drugs, theoretically allows for low concentrations of the antisense drug to be used as compared to conventional drugs. The simplicity of this concept has led to the use of ASO drugs as therapeutic agents. The use of conformationally restricted nucleotides as antisense modifications has led to an impressive progress in the field of oligonucleotide therapeutics. In particular, Locked Nucleic Acids (LNAs) have been outlined as a very promising antisense modification due to their unparalleled hybridization properties. Thus, LNA has become the focus of multiple research groups and pharmaceutical companies, leading to the development of LNA modified ON candidates.
During my Ph.D. work, I developed numerous chemically modified LNA building blocks by using nucleobase functionalization strategy to modulate the biophysical properties of respective building block. We conducted studies on different nucleobases (cytosine and adenosine) where we have shown that LNA building blocks, which are modified at the C5 position of cytosine pyrimidines and C8 position of adenine purines. We also studied the properties of C5 LNA (U) monomers conjugated to bulky hydrophilic carbohydrates substituents. The goal of the study was to develop nucleotides that confer stability towards exonuclease enzymes without disrupting the duplex stability as hydrophobic substituents.
Chemically modified oligonucleotides are finding an increasing use in diagnostics as fluorescent hybridization probes to detect DNA mutations and to study DNA-protein interactions. We have developed DNA probes centrally modified with 5-[3-(1-pyrenecarboxamido) propynyl] DNA monomer, which are flanked with conventional LNAs as diagnostic probes for detection of single nucleotide polymorphism in DNA. These neighbouring LNAs influence the position of the fluorophore in a similar manner as for the C5-fluorophore functionalized LNA probes. These probes are relatively easier to synthesize and results in greater fluorescence based discrimination of matched vs mismatched duplexes than just C5-LNA or C5-DNA fluorophore functionalized probes.
These studies conducted on Locked Nucleic Acids (LNAs) suggested, that, i) nucleobase functionalization improves the therapeutic properties of LNA; ii) introduction of canonical LNAs in an ON sequence can be used to develop diagnostic probes with improved photophysical properties.Thesis (Ph.D., Chemistry)--University of Idaho, June 201
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Invader Probe Targeting of Chromosomal DNA
No full textInterest in developing probes capable of targeting chromosomal DNA in cells has grown to meet diagnostic and therapeutic needs. DNA has a stable predicable double-stranded structure that has been the subject of study to identify agents that can specifically bind to the duplex (CHAPTER 1). Success stories from probe technologies such as triplex-forming oligonucleotides, peptide nucleic acids (PNAs), and minor-groove-binding polyamides have been well characterized. However, they suffer limits of detection with experimental conditions requirements (homopurine targets; denaturing steps; low ionic strengths; short target sequences). Newly discovered CRISPR-Cas9 has garnered much attention; however, the approach requires transfection of plasmids encoding CRISPR-Cas9 components. To address these shortcomings, our laboratory has developed Invader probes. Placement of 2'-O-(pyren-1-yl)methyl RNA monomers in +1 interstrand zipper arrangements destabilizes the probe duplex as the intercalating pyrene moieties vie for the same space between two Watson-Crick base pairs. These ‘energetic hotspots’ activate the double-stranded probe and, in concert with the high affinity for complementary DNA (cDNA) displayed by individual probe strands, provide the driving force for recognition of mixed-sequence target sites. The capabilities and features of Invader probes specific detection of chromosomal DNA fluorescent in situ hybridization (FISH) assays at near physiological conditions were statistically analyzed. Based on these results, Optimized Invader probes were synthesized and showed improved efficiency in chromosomal DNA detection (CHAPTER 2). Moreover, the combination of Invader FISH probes with the powerful detection properties of flow cytometry was used to quantify thousands of specifically label isolated nuclei, offering a potential advantage over the laborious microscope evaluation of detection (APPENDIX A).masters, M.S., Chemistry -- University of Idaho - College of Graduate Studies, 2020-0
Exploiting the Energy of Intercalation for Sequence-Specific Recognition of Double-Stranded DNA
No full textThere has been great interest in developing probes capable of recognizing specific regions of double-stranded DNA (dsDNA) due to the vast potential of applications in the fields of molecular biology, genomic diagnostics, and DNA nanotechnology. Early efforts focused on targeting the accessible grooves of the DNA helix by means of triplex forming oligonucleotides (TFOs) or pyrrole/imidazole polyamides. However, such attempts have been limited by sequence restrictions and/or requirement for non-physiological conditions. A conceptually more elegant, but also more challenging, approach is to target the buried base pairs of the duplex and utilize the intrinsic Watson-Crick binding rules of nucleic acids. Single-stranded peptide nucleic acids (PNA) have a neutral backbone and bind to complementary DNA with very high affinity, facilitating duplex invasion, albeit at low ionic strengths. We have introduced Invader probes as an alternative approach for recognition of dsDNA. A mini-review of this approach is presented in CHAPTER 1. These probes are modified double-stranded oligonucleotides that are energetically activated for dsDNA recognition due to the low stability of the probe duplex and high affinity duplexes of each individual probe strand towards complementary dsDNA. The driving force for dsDNA recognition is the result of 2’-intercalator-functionalized nucleotides positioned in +1 interstrand zipper arrangements, termed ‘energetic hotspots’. Major efforts in the laboratory have been focused on optimizing the chemistry of the monomers, while my projects primarily focused on the optimization of the probe architecture and applications of Invader probes. Initially, I set out to determine the influence of the number, location, and distance between the energetic hotspots. Optimal probe designs were then used for sequence-specific recognition of chromosomal DNA in a non-denaturing fluorescence in situ hybridization assay (CHAPTER 2). I then introduce bulged Invader probes, which feature an alkyl bulge in the double-stranded probe, which induces fraying and localized perturbation, resulting in faster and more efficient recognition of dsDNA (CHAPTER 3 and 4). Invader probes capable of photoactivated interstrand cross-linking are presented in APPENDIX A, which results in a covalent bond between the probe and DNA strands, resulting in more persistent binding. Finally, I evaluated Invader probes for inhibition of in vitro transcription (APPENDIX B).doctoral, Ph.D., Chemistry -- University of Idaho - College of Graduate Studies, 201
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Invader-mediated Targeting of Chromosomal DNA
No full textInterest in developing probes capable of targeting chromosomal DNA in cells has grown to meet diagnostic and therapeutic needs. DNA has a stable, predicable double-stranded structure that has been the subject of study to identify agents that can specifically bind to the duplex (CHAPTER 1). Success stories from probe technologies such as triplex-forming oligonucleotides, peptide nucleic acids (PNAs), and minor-groove-binding polyamides have been well-characterized. However, they suffer limits of detection along with challenging experimental conditions requirements (homopurine targets; denaturing steps; low ionic strengths; short target sequences). The newly discovered CRISPR-Cas9 has garnered much attention. However, the approach requires transfection of plasmids encoding CRISPR-Cas9 components. To address these shortcomings, our laboratory has developed Invader probes. Placement of 2'-O-(pyren-1-yl)methyl RNA monomers in +1 interstrand zipper arrangements destabilizes the probe duplex as the intercalating pyrene moieties vie for the same space between two Watson-Crick base pairs. These ‘energetic hotspots’ activate the double-stranded probe and, in concert with the high affinity for complementary DNA (cDNA) displayed by individual probe strands, provide the driving force for recognition of mixed-sequence target sites. The capabilities and features of Invader probes for specific detection of chromosomal DNA in fluorescent in situ hybridization (FISH) assays at near physiological conditions were statistically analyzed. Based on these results, optimized Invader probes were synthesized and showed improved efficiency in chromosomal DNA detection (CHAPTER 2). Moreover, the combination of Invader FISH probes with the powerful detection properties of flow cytometry was used to quantify thousands of specifically labelled isolated nuclei, offering a potential advantage over the laborious microscope evaluation of detection (CHAPTER 3).masters, M.S., Chemistry -- University of Idaho - College of Graduate Studies, 2020-0
- …
