The Discovery and Characterization of Potential Anti-Gene Enhancers
Poster Number
4A
Introduction/Abstract
Flavonoids are a large class of plant pigment compounds that have been shown to have many effects on the body, including anti-inflammatory, antioxidant, and modulating many key cellular enzymes. One of the most abundant and highly researched compounds is Quercetin. Quercetin has been used as a homeopathic treatment and prevention for cardiovascular disease, allergy relief, rheumatic diseases, and cancer. Although marketed as a dietary supplement, Quercetin is ingested through a normal diet consumed through vegetables, fruits, teas, and wine. Quercetin has only been shown to interact with the DNA duplex at high concentrations (> 250μM); therefore, our group has investigated the effects of newly synthesized Quercetin derivative on DNA and RNA duplexes and triplexes to understand further therapeutic uses.
Purpose
Given the knowledge of Quercetin’s ability to modulate many key enzymes in the body, our group took an interest in investigating the binding of newly synthesized derivatives to DNA and RNA. Characterizing their binding affinity to triplex DNA allows the understanding of how further modification may enhance DNA triplex affinity for the potential use of anti-gene therapy enhancement.
Method
General Material and Methods:
All the chemicals were purchased from MilliporeSigma or Fisher Scientific and used without further purification. DNA oligonucleotides were purchased from Fisher Scientific. Polynucleotides were purchased from MilliporeSigma. The concentrations of polynucleotide solutions were determined spectrophotometrically using the following extinction coefficients (in units of mol of nucleotide/L-1 cm-1). The concentrations of DNA solutions were determined spectrophotometrically, using the molar extinction coefficients (in units of mol of strand/L-1 cm-1) obtained from OligoAnalyzer (www.idtdna.com). UV spectra were recorded on a Varian Cary 100 Bio UV-Vis spectrophotometer equipped with a thermoelectrically controlled 6 × 6 cell holder. Circular dichroism (CD) spectra were recorded on a JASCO J-810 spectropolarimeter using a quartz cuvette with a 1 mm or 1 cm optical path length. Isothermal microcalorimetric measurements were performed on a TA Instruments Affinity ITC LV. DSC thermograms were recorded on a TA Instruments Nano DSC.
DNA Preparation for UV and CD
15uM or 1uM of either polynucleotide or oligonucleotide, respectively, were mixed in 10 mM sodium cacodylate buffer and 150 mM KCl or 100 mM NaCl, respectively, at pH 7, heated to 95 °C for 5 min, slowly cooled down to room temperature and stored at 4 °C overnight.
Thermal Denaturation Monitored by UV
Using the UV-Vis spectrophotometer, the UV absorbance at 260 nm was recorded as a function of temperature from 25-90 ̊C or 5-90 ̊C at a 0.5 ̊C/min scan rate. The melting temperature (Tm) was determined using the first derivative method, which is defined as the temperature at which 50% of the DNA is dissociated into random coils.
CD Titration
Using a polynucleotide triplex solution prepared similarly to the UV samples, aliquots of the selected ligand were gradually added and incubated for 5 min before collecting an average of 3 spectra from 200-700nm at a scan speed of 100 nm/min, and the data pitch was 0.5 nm.
Isothermal Titration Calorimetry (ITC)
After dialysis of a 10μM oligonucleotide sample in the sample conditions as UV, heat burst curves were generated using ITC and analyzed using the NanoAnalyzer software. The heat associated with ligand-DNA binding was obtained by subtracting the heat associated with ligand-buffer injections. Typically, 5μL of a 100 μM ligand solution were injected into 185μL of either DNA or buffer solution at a temperature of 15˚C.
Differential Scanning Calorimetry (DSC)
After dialysis, two cells, one containing 600 μL of 100 μM polynucleotide in the absence or presence of a ligand and the other filled with an equal volume of the buffer, were heated from 25 °C to 90 °C at a rate of 0.2 °C/min. Thermodynamic parameters were obtained with NanoAnalyze software using the Gaussian model. The buffer conditions were the same as the UV preparation.
Results
Our group has discovered a novel class of DNA specific triplex binding ligands. Our data show comparable DNA triplex stabilization to a known triplex binding ligand without any effect on the DNA duplex or RNA duplex and triplex structures.
Significance
These findings demonstrate the significance of modification positioning around the Quercetin structure. The modification positions of these derivatives allow for selective binding and stabilization of DNA triplexes without any effect on native DNA duplexes or RNA structures. This provides a highly specific enhancement interaction for anti-gene therapeutic use.
Location
William Knox Holt Memorial Library and Learning Center, University of the Pacific, 3601 Pacific Ave., Stockton, CA 95211
Format
Poster Presentation
Poster Session
Morning
The Discovery and Characterization of Potential Anti-Gene Enhancers
William Knox Holt Memorial Library and Learning Center, University of the Pacific, 3601 Pacific Ave., Stockton, CA 95211
Flavonoids are a large class of plant pigment compounds that have been shown to have many effects on the body, including anti-inflammatory, antioxidant, and modulating many key cellular enzymes. One of the most abundant and highly researched compounds is Quercetin. Quercetin has been used as a homeopathic treatment and prevention for cardiovascular disease, allergy relief, rheumatic diseases, and cancer. Although marketed as a dietary supplement, Quercetin is ingested through a normal diet consumed through vegetables, fruits, teas, and wine. Quercetin has only been shown to interact with the DNA duplex at high concentrations (> 250μM); therefore, our group has investigated the effects of newly synthesized Quercetin derivative on DNA and RNA duplexes and triplexes to understand further therapeutic uses.
