Title

Flavone Derivatives: Novel Ligands for Triplex Binding

Poster Number

5B

Lead Author Affiliation

Chemical Synthesis, Drug Discovery and Design

Lead Author Status

Doctoral Student

Second Author Affiliation

Chemical Synthesis, Drug Discovery and Design

Second Author Status

Doctoral Student

Third Author Affiliation

Chemical Synthesis, Drug Discovery and Design

Third Author Status

Doctoral Student

Fourth Author Affiliation

Chemistry

Fourth Author Status

Faculty

Introduction

Triplex DNA forms upon the sequence-specific binding of a third strand into the major groove of a duplex DNA. Triplexes that form in living organisms are known as H-DNA, and they can be found in promoter regions, spliceosome complexes, and telomeric complexes. They are also implicated in genetic diseases such as tuberculosis sclerosis complex, autosomal dominant polycystic kidney disease, and Friedreich’s ataxia. Because of its high specificity, triplex DNA makes an ideal target for antigene therapy. Normal cellular machinery is unable to recognize triplex DNA, so we can stop the production of proteins. However, triplexes form much slower than duplexes, and triplexes are less stable than duplex DNA under physiological conditions. There have been many approaches to try and stabilize triplexes, such as making modifications to the TFO backbone, modifications of the nucleobases, and the use of small molecules.

Purpose

Recently, our group has discovered a class of flavonoid derivatives that have a strong stabilizing effect on triplexes but show no effect on duplexes. Herein, we report further structural variations of flavonoid derivatives that have an easier synthesis, which will allow for mass scale synthesis. We hope that these new compounds will specifically bind to the triplexes to stabilize them and not bind to duplexes.

Method

Novel flavonoid derivatives were synthesized. The resulting flavone-based derivatives are subject to various biophysical studies including thermal denaturation monitored by UV and microcalorimetry.

In thermal denaturation monitored by UV, solutions of the DNA or RNA and the ligand in buffer were made in duplicate. In the control experiment, only DNA was added. Either 15 mM of the polynucleotide or 1 µM of the oligonucleotide 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 minutes and slowly cooled back down to room temperature before storing at 4 ˚C overnight. The resulting solutions were heated either from 5 ˚C or 25 ˚C to 90 ˚C at a rate of 0.2 ˚C/min. They were monitored at 260 nm. The resulting curves were replotted in Origin, and the first derivative was used to determine the melting temperature. The melting temperature is defined at which 50% of the DNA is dissociated into random coils.

In the microcalorimetry study, heat change was measured as a solution of ligand was slowly added to a solution of DNA. Solutions of DNA were dialyzed to remove any impurities and proteins. A 100 μM solution of ligand was injected into a 10 μM solution of dialyzed DNA. Heat burst curves were generated using ITC and analyzed using the NanoAnalyze software. An experiment of ligand titrated into buffer was also performed to subtract any heat associated from the ligand titrating into the solution of buffer. Typically, injections of 5 µL were injected into 185 μL of DNA or buffer at a temperature of 15 ˚C. Using an independent model, we were able to calculate the thermodynamic parameters, such as ΔH and ΔS as well as the dissociation constant, Kd.

Results

The newly synthesized compounds show an increased melting temperature of the DNA triplex, very much similar to the lead compounds. They also show no effect on duplexes or RNA triplexes. Furthermore, the side chain at the 5-position is important for triplex binding. The Kd obtained from the microcalorimetry experiment was comparable to a well-known triplex DNA binding ligand, neomycin.

Significance

We have successfully synthesized a simpler class of compounds that will specifically target triplex DNA and not duplex DNA. Using these compounds, we hope to stabilize triplexes in living organisms and potentially stop the production of proteins that can cause disease.

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

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Apr 30th, 10:00 AM Apr 30th, 12:00 PM

Flavone Derivatives: Novel Ligands for Triplex Binding

William Knox Holt Memorial Library and Learning Center, University of the Pacific, 3601 Pacific Ave., Stockton, CA 95211

Triplex DNA forms upon the sequence-specific binding of a third strand into the major groove of a duplex DNA. Triplexes that form in living organisms are known as H-DNA, and they can be found in promoter regions, spliceosome complexes, and telomeric complexes. They are also implicated in genetic diseases such as tuberculosis sclerosis complex, autosomal dominant polycystic kidney disease, and Friedreich’s ataxia. Because of its high specificity, triplex DNA makes an ideal target for antigene therapy. Normal cellular machinery is unable to recognize triplex DNA, so we can stop the production of proteins. However, triplexes form much slower than duplexes, and triplexes are less stable than duplex DNA under physiological conditions. There have been many approaches to try and stabilize triplexes, such as making modifications to the TFO backbone, modifications of the nucleobases, and the use of small molecules.