Browsing by Author "Shaw, Barbara R"
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Item Open Access Biophysical Investigations of Boranophosphate siRNA for Use in RNA Interference against Human Disease(2009) Moussa, LauraThis project is predicated on the ability of the boranophosphate modification of siRNA to increase its therapeutic applicability for gene silencing in in vitro and in vivo systems. It has been shown that the boranophosphate (BH3-PO3) can overcome many of the limitations that are traditionally found when using RNAi, namely nuclease stability. The synthesis of siRNA modified with 5'-(alpha-P-borano)-nucleoside triphosphates (NTP) analogs alone and in combination with 2'-deoxy-2'-fluoro nucleoside triphosphate analogs were performed and optimized. It was found that normal RNA transcriptions showed the highest yield with higher NTP concentrations and shorter incubation times. Boranophosphate modified RNA and 2'F/borano modified RNA transcription yield was optimal at lower NTP concentrations and extended incubations. The boranophosphate NTPs and RNA were characterized with high performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance, indicating successful synthesis of NTPalphaB and 2'F NTPs. PAGE and mass spectrometry analysis were performed to ensure full-length transcription of the modified siRNA molecules. The effects of these modifications were explored with respect to the biophysical properties of the modified homoduplex and heteroduplex siRNA. The techniques used in this work included hybridization affinity assays (melting temperature), secondary structure determination (circular dichroism), nuclease stability assays, and assessment of the lipophilicity of the modified siRNA by determining partition coefficients.
Modification of siRNA with boranophosphate and 2'fluoro/borano modified NTPs appears to have caused the homoduplexes and heteroduplexes to adopt a more B form-like helix that had lower Tm compared to unmodified RNA. The stability of the siRNA transcript to enzymatic hydrolysis by Exonuclease T was on the order of 2'fluoro/borano> normal = boranophosphate. Boranophosphate modification increased the stability of the transcript to enzymatic hydrolysis by the endonuclease RNase A, compared to both normal and 2' fluoro modified siRNA. Overall, the 2' fluoro/borano modified siRNA showed the greatest biological stability. Modification of the siRNA with increasing percentages of boranophosphates resulted in increasing lipophilicity of the molecule up to 60-fold, compared to both normal and 2' fluoro RNA.
A method to site-specifically modify the boranophosphate siRNA using T4 RNA ligase was also investigated. Finally, the siRNA in this work was tested in several in vitro systems, yielding promising results for the usage of boranophosphate siRNA for use against human viruses and cancers. It was shown that in for in vitro systems for human papillomavirus gene expression (HeLa, SiHa, and W12E) and luciferase expression (B16F10 cells), boranophosphate modified siRNA can specifically downregulate gene expression, and in the case of human papillomavirus, can downregulate cell growth.
Item Open Access Boranophosphate-Modified Nucleic Acids as Biomolecular Probes: Synthesis, Substrate, and Antiviral Properties(2011) Cheek, Marcus AdrianIn boranophosphate (BP) nucleotides, a borane (BH3) group is substituted for a non-bridging phosphoryl oxygen of a normal phosphate group, resulting in a class of modified isoelectronic DNA and RNA mimics that can modulate the reading and writing of genetic information. 5'-(α-P-borano)nucleoside triphosphates (NTPαBs) are good substrates or inhibitors for many viral RNA polymerases and reverse transcriptases (RT) when compared with natural nucleoside 5'-triphosphates (NTPs). A practical aspect of this coding phenomenon employs T7 bacteriophage DNA-dependent RNA polymerase (DdRP) to synthesize BP-modified RNA utilizing NTPαBs as monomeric substrates. Additionally, the α-P-borano modification can be used to probe the catalytic phosphoryl transfer mechanism used by viral polymerases, and possibly enhance existing anti-viral chain terminating nucleotides.
The primary goal of this dissertation is to better understand the effects of NTPαBs on the activity of viral polymerases. In the last decade several NTPαBs have been shown to be efficient and selective substrates for wild-type (wt) and, to a greater extent, HIV and MMLV drug-resistant viral reverse transcriptases. More recently NS5B, the Hepatitis C viral RNA-dependent RNA polymerase (HCV RdRP), is a viable target for nucleotide-based inhibition studies. Due to the similarities between the active sites of HIV-RT and HCV NS5B, it is therefore relevant to investigate the substrate properties of this unique modification. We investigated, for the first time, the inhibition kinetics of HCV NS5BΔ55 RdRP by two newly synthesized NTPαB analogs: 2'-O-methyladenosine 5'-(α-P-borano) triphosphate (2'-OMe ATPαB, 9a) and 3'-deoxyadenosine 5'-(α-P-borano) triphosphate (3'-dATPαB, 9b) and the steady state incorporation kinetics of ATPαB (51a). Our results showed that:
(1) Rp-2'-OMe ATPαB (9a) and Rp-3'-dATPαB (9b) exhibited a 3.5- and 16-fold lower IC50 respectively when compared with natural phosphate controls, suggesting greater inhibitory potency.
(2) Additionally 9a and 9b demonstrated a 5- and 21-fold lower inhibition constant (Ki) respectively when compared with the natural phosphate. Both compounds retained the competitive inhibition behavior of their parent nucleotides.
(3) HCV NS5BΔ55 preferred the Rp isomer of ATPαB (Vmax/Km = 0.095) over the natural ATP substrate (Vmax/Km = 0.057). None of the Sp isomers were substrates for HCV NS5BΔ55. We further concluded that wild-type (wt) HCV NS5B seems to discriminate against 3'-deoxy NTPs via lost interactions between the 3'-OH on the ribose and the active site residues, or lost intramolecular hydrogen bonding interactions between the 3'-OH and the pyrophosphate leaving group during phosphoryl transfer. The overall implications of this proof of concept study are that existing viral RdRP inhibitors could be retro-fitted with the boranophosphate modification to possibly increase potency.
This dissertation also explored the synthesis of anti-HIV-RT boranophosphate nucleotides which act through a chain terminating or mutagenic mechanism. 2'-3'-didehydro-2'-3'-dideoxythymidine 5'-(α-P-borano)-diphosphate (D4TDPαB, 30) was synthesized and later stereoselectively phosphorylated to yield the Rp-form of D4TTPαB (31). This was tested as a substrate in two multi-drug resistant forms of HIV-RT. Additionally, the NTPαB analogue of the mutagenic 5-aza-5,6-dihydro-2'-deoxycytidine (KP-1212-TPαB, 16) was synthesized with the eventual goal of inducing error catastrophe during viral genomic replication.
Lastly we detail the extraction and purification of gemcitabine (dFdC) from Gemzar® drug mixture using a derivatization method that produced a protected form of gemcitabine nucleoside. This protected gemcitabine was then used to synthesize gemcitabine 5'-triphosphate (dFdCTP, 42).
Item Open Access Exploring the Immunogenicity and Therapeutic Applications of Boranophosphate-modified RNA: siRNA and RNA Aptamers(2011) Sharaf, Mariam LucilaBorane (BH3) chemistry offers unique chemical characteristics that enable boranophosphate (BP) oligonucleotides with potential to enhance RNA therapeutic applications such as RNA interference (RNAi) and RNA aptamers. Further, BP nucleotides are substrates for RNA polymerases which allow the enzymatic synthesis of stereoregular boranophosphate (BP)-RNA molecules of different lengths and properties. We expect that these BP-RNAs will interact in a novel way with the desired target molecules because they can coordinate with a diverse array of ligand sites in proteins or other RNA molecules. This is due to the distinct hydrophobicity, sterospecificity, and polarity properties imparted by the phosphorus-boron (P-B) chemical bond compared to the natural phosphorus-oxygen (P-O) bond.
The object of this dissertation is to explore the therapeutic applications of the BP-RNA such as siRNA, RNA aptamers, and in addition investigate the immunogenicity of this modification. We used mouse cells to determine if BP-RNA would activate toll-like receptor (TLR 7), which is involved in innate immune response to foreign single stranded RNA (ssRNA). This response is undesired when applied to oligonucleotide therapeutics such as siRNA and RNA aptamers. In terms of RNAi, it would be an advantage to have low immunogenicity and high downregulation activity by the siRNA. To determine the innate immune activation of the BP-RNA through the TLR 7 we used a known activator, the human immunodeficiency virus (HIV) derived single-stranded RNA (ssRNA40) and measured the production of cytokines as a function of the number of modified BP-linkages. The production of cytokines IL-6 and TNFα was quantified after the boranophosphate (BP), phosphorothioate (PS) or natural ssRNA40 were transfected into murine macrophage Raw264.7 cells. Natural and phosphorothioate RNA (PS-RNA) have been shown to be activators of TLR 7 receptors. In contrast, we found that fully modified BP- ssRNA40 did not activate TLR 7. This is relevant in oligonucleotide applications such as siRNA and RNA aptamers where off-target effects such as immune activation after administration are not desired.
Subsequently, the low immune activation would be an advantage when coupled to RNAi activity of the oligonucleotide. Thus, we explored whether BP modified siRNA molecules would modulate gene expression and if there was an effect on downregulation activity when increasing the number of BH3 modifications on the phosphate backbone. Our therapeutic model was the multi-drug resistance 1 (MDR1) gene that expresses P-glycoprotein (P-gp), which has been notoriously difficult to modulate. The aberrant regulation of genes such as MDR1 in cancer cells are a major cause of chemotherapeutic treatment failure against human cancers. Hence, controlling the expression of cancer genes with antisense technology is a possible cancer therapy. Specifically, correcting the overexpression of p-glycoprotein using modified siRNAs that target and degrade the P-glycoprotein mRNA produced by the MDR1 gene. We found that there is a reduction of siRNA activity with an increasing number of BP-modifications. It appears that there is a fine balance between lack of immune response and gene downregulation when applied to BP-siRNA.
Finally, we compared the enrichment during the Systematic Evolution of Ligands by EXponential enrichment (SELEX) method of two libraries, one BP-RNA (UαB) compared to a doubly-modified RNA (2'FC & UαB), against a human thrombin. Aptamers modulate protein activity and interfere with protein signaling by binding to the desired protein with high affinity and specificity leading to their use in therapeutic applications where protein activity needs to be controlled or it is anomalous. In the case of blood coagulation, thrombin plays a central role in coagulation signaling cascade and it is a good target to use to control blood coagulation in clinical settings. We attempted to optimize the selection of BP- RNA aptamers through 4-8 rounds of SELEX against the protein thrombin. We found that the selection conditions were not optimal for BP-RNA SELEX possibly due to non-specific binding to a bovine serum albumin (BSA) in the selection buffer.