Oligonucleotides (ONs) are typically defined as short nucleic acid polymers that are 20 to 200 bases long. It has been well known that aptamers (single-stranded DNA or RNA ONs) can adopt distinct three-dimensional folded structures and bind to target proteins (or other target molecules) with high affinity and selectivity. While aptamers that target proteins represent a promising molecular recognition modality that exploits the self-folding nature of nucleic acids, alternative protein recognition elements, usually synthetic small molecules that are projected from an ON scaffold, can also be utilized for protein-binding purposes. In particular, ONs tethered to protein-binding small organic molecules have received recent attention due to the capability of synthetic molecules to serve as specific inhibitors of proteins associated with disease and so these synthetic moieties can complement or augment the molecular recognition capacity of ONs. Further, the ON domain can also (a) serve as a “barcode” to identify the synthetic fragments and (b) act as a scaffold to project them in a desired and programmable fashion. This dissertation starts with representative examples, wherein ONs serve as binding moieties and/or as projecting scaffolds for synthetic protein-recognition elements, discussed in chapter one, followed by the development of two novel ON-based protein inhibitors which further explore the approach of coupling synthetic protein-binding fragments with ON scaffolds. The first system, as described in chapter two, exhibits controllable protein inhibition against the target protein in selective response to a cancer-associated microRNA. The second system (explored in chapter three) features an aptamer core sequence flanked by small protein-binding elements. The resultant aptamer chimera is capable of forming a complex simultaneously with two target proteins, leading to dual inhibition. / acase@tulane.edu

Oligonucleotides are synthetic strings of DNA or RNA used mostly for biochemical analysis and diagnostics. For them to be useful in these fields, a purity over 90% is most often required. However, when synthesizing these sequences, many “failures” (shorter sequences) are made in the step-wise process. The synthesized oligonucleotides need to therefore be purified. This is most often done with gel electrophoresis or liquid chromatography. These methods are, on the other hand, very time-consuming and laborious. Solid phase extraction (SPE) is a much faster purification method if optimized and it can be done with the standard cartridges as well as 96-well plates, that allow many samples to efficiently be run at the same time. With reversed phase (RP) SPE, the dimethoxytrityl (DMT) group, that is attached to the target at the final synthesis step, can be used for stronger retention to the bed sorbent and leaving only the target at the final eluting stage. The impurities without a DMT-on group, that do not adsorb to the sorbent, are washed away in earlier steps. The purpose of this study is to optimize an SPE method for purification of oligonucleotides. Two different cartridges, Clarity QSP (Phenomenex) and Glen-Pak (Glen Research) were used. The purity analysis and oligonucleotide identification were done using anion exchange - high performance liquid chromatography (AIE-HPLC) and time-of-flight mass spectrometry (TOF MS). To conclude, Clarity QSP achieved, at the most, a purity of 68.8% with the recommended SPE steps by Phenomenex. Alterations in the extraction procedure resulted in similar purity or lower. Glen-Pak reached a peak purity of 78.8% when doing a double salt wash of 5% ACN in 2 M sodium chloride and another double wash after detritylation with 1% acetonitrile. This method has to be further optimized in order to reach a purity of at least 90% to be useful in industrial settings.

There are different kinds of recognition molecules that specifically can detect and bind target molecules. Antibodies, with their two light and two heavy chains can detect and bind any kind of antigens. Molecular imprinting is a technology to prepare specific polymers that selectively bind target molecules. The technology has received wide attention in recent years because it provides a viable method for creating a polymer that is complementary in shape and binding sites to a template. The synthesized polymer is called a molecularly imprinted polymer (MIP) or a plastibody. Molecular imprinting shows promise in diverse areas as chromatography, antibody mimics, solid phase extraction and more. An alternative to molecular imprinting and other types of recognition has recently been postulated where DNA polymers based on functionalized polynucleotides build up a polymer network that are able to specifically recognize a target. This approach is characterized by binding of oligonucleotides carrying functional organic groups, to the target molecule followed by connecting the functionalized oligonucleotides forming a DNA polymer that specifically recognizes the target. The polymer is called a DNAbody. Thus, a DNAbody is a polymer structure based on DNA conjugated with functional organic groups which specifically can detect and bind to a template. The DNA-bodies can be copied and produced in larger amounts by PCR. This study showed that at least one functional oligonucleotide was able to interact with the target antibody. It was also seen that some interaction occurred between the DNA and protein. The results also indicated that it is possible to perfom DNA polymerase reactions in presence of an antibody.

The technique of DNA origami utilizes the specific and limited bonding properties of DNA to fold single stranded DNA sequences of various lengths to form a predesigned structure. One longer sequence is used as a scaffold and numerous shorter sequences called staples, which are all complementary to the scaffold sequence, are used to fold the scaffold into intricate shapes. The most commonly used scaffold is derived by extracting the genome of the M13 phage and the staples are usually chemically synthesized oligonucleotides. Longer single stranded sequences are difficult to synthesize with high specificity, which limits the choices of scaffold sequences available. In this project two main methods of single stranded amplification, Rolling Circle Amplification (RCA) and the usage of helper phages, were explored with the goal to produce both a 378 nt scaffold and staple sequences needed for folding a DNA origami structure. To facilitate imaging by Transmission Electron Microscopy (TEM) of this small structure, the DNA origami structure was created to form a polymer structure. Production of the scaffold sequence in high yield was unsuccessful and no well-defined polymers were found in the folded samples, though a few results showed promise for further studies and optimizations. Due to time constraints of this project, only production of the scaffold sequence was tested. Unfortunately the scaffold produced by the helper phages was of the complementary strand to that used to design the DNA origami structure, and could therefore not be used for folding. The correct strand was produced by the RCA where the yield was too low when using Phi29 DNA polymerase for proper folding to take place, though small scale RCA by Bst DNA polymerase on the other hand showed promising results. These results indicate that the scaffold production may not be far off but still more experience in producing intermediate size oligonucleotides may be necessary before succeeding in high yield production of this 378 nt long sequence. The promise given by this production is to enable high yield, high purity, low cost and also an easily scalable process set-up. This would be an important step in future DNA nanotechnology research when moving from small scale laboratory research to large scale applications such as targeted drug delivery systems.

DNA condensation is a fundamental process in all living organisms. The highly abundant nucleoid-associated proteins, HU and IHF, present in bacteria, have been shown to play an important role in shaping the nucleoid. However, the exact mechanism is not well understood. In this thesis, we have demonstrated that both HU and IHF guide DNA to condense into linear bundle-like structures in presence of cellular condensing components, but the proteins alone do not condense DNA into densely packed structures. Our results suggest a mechanism by which HU and IHF could act as architectural proteins during in vitro and in vivo DNA condensation. More recently, DNA condensation has attracted much attention for its relevance in optimizing artificial DNA delivery systems for gene therapy. The research presented in this dissertation provides in depth biophysical studies that demonstrate how local modulations in the nucleic acid structure can be used to control both the size and the morphology DNA condensates. We describe a novel strategy for improving the condensation of oligonucleotides that is based on the self-organization of half-sliding complementary oligonucleotides into long duplexes (ca. kb) with flexible sites at regular intervals along the duplex backbones, in the form of single-stranded nicks or single-stranded gaps. Our results also provide new insights into the role of DNA flexibility in condensate formation and suggest the potential for the use of this DNA structure in the design of vectors for oligonucleotide and gene delivery.

Thesis (Ph. D.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains xii, 161 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 140-158).

Thesis (Ph. D.)--West Virginia University, 2003. / Title from document title page. Document formatted into pages; contains xiii, 180 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 138-151).

In vitro, G-rich sequences form highly stable secondary structures known as G-Quadruplexes. These structures have been characterized by circular dichroism nuclear magnetic resonance and X-ray crystallography; although their detection in vivo has remained elusive. Due to the biological implication of a transisent and polymorphic secondary structure forming within the hypothetical G-Quadruplex forming regions, there is growing interest to understand their in vivo molecular dynamics. / text

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Regulation of biological processes through the use of genetic elements is a central part of biological research and also holds great promise for future therapeutic applications. Oligonucleotides comprise a class of versatile biomolecules capable of modulating gene regulation. Gene therapy, the concept of introducing genetic elements in order to treat disease, presents a promising therapeutic strategy based on such macromolecular agents. Applications involving charged macromolecules such as nucleic acids require the development of the active pharmaceutical ingredient as well as efficient means of intracellular delivery. Cell-penetrating peptides are a promising class of drug delivery vehicles, capable of translocation across the cell membrane together with molecules otherwise unable to permeate cells, which has gained significant attention. In order to increase the effectiveness of cell-penetrating peptide-mediated delivery, further understanding of the mechanisms of uptake is needed in addition to improved design to make the cell-penetrating peptides more stable and, in some cases, targeted. This thesis encompasses four scientific studies aimed at investigating cell-penetrating peptide and oligonucleotide designs amenable to therapeutic applications as well as elucidating the mechanisms underlying uptake of cell-penetrating peptide:oligonucleotide nanoparticles. It also includes an example of a therapeutic application of cell-penetrating peptide-mediated delivery of oligonucleotides. Paper I presents a study evaluating a range of chemically modified anti-miRNAs for use in the design of therapeutic oligonucleotides. All varieties of oligonucleotides used in the study target miRNA-21 and are evaluated using a dual luciferase reporter system. Paper II introduces a novel cell-penetrating peptide, PepFect15, aiming at combining the desirable properties of improved peptide stability and efficient cellular uptake with a propensity for endosomal escape, to produce a delivery vector well suited for delivery of oligonucleotides. The performance of this new cell-penetrating peptide was evaluated based on its delivery capabilities pertaining to splice-correcting oligonucleotides and anti-miRNAs. Paper III investigates the involvement of scavenger receptor class A in the uptake of various cell-penetrating peptides together with their oligonucleotide cargo. Finally, paper IV aims at using cell-penetrating peptide-mediated delivery to improve the efficiency of telomerase inhibition by antisense oligonucleotides targeting the telomerase enzyme ribonucleotide component. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>