Part One: Synthesis and applications of endcapped oligonucleotides. Part Two: Synthesis and cellular delivery of amine and guanidinium functionalized gold nanoparticles

Pei-Sze Ng, Purdue University

Abstract

Part 1. Endcaps can be visualized as linkers that link the 3' and 5' ends of DNA duplexes. A series of hydrophilic diamide endcaps has been synthesized and shown to stabilize very short DNA duplexes (4 base-pair) with Tm values in the range of 42-55 °C. Endcapped CpG oligonucleotides consisting of single-stranded, nicked double-stranded and covalently closed double-stranded forms were synthesized to investigate their immunostimulatory activities and to elucidate the mode of recognition of different forms of CpG oligonucleotides by the immune system. Endcaps were incorporated into both the 3' and 5' ends of self-complementary DNA duplexes (8-20 base-pair) to form nicked, DNA duplexes that were capped on both ends. Closed endcapped DNA duplexes were obtained by enzymatic ligation of the nicked DNA duplexes. Analysis of the ligation mixtures by HPLC and PAGE indicated that endcapped duplexes smaller than 11 base-pair were not ligated. In addition, the efficiency of ligation was dependent on the number of nucleotides flanking the 3'- and 5' ends of the nick. The nuclease stability of these endcapped CpG oligonucleotides was established to be at least 6-fold more stable than unmodified oligonucleotides, with half-lives of 30 minutes for the endcapped single stranded DNA and 6-20 hours for the ligated and nicked endcapped DNA duplexes, respectively. Endcapped CpG oligonucleotides stimulated IL-6 and IL-12 secretion (in vitro) and were administered to tumor implanted mice. The nicked endcapped DNA duplexes and the endcapped single stranded DNA extended the life span of the mice beyond that of the untreated control mice. Part II. Thiol terminated amine and guanidinium derivatives were synthesized on solid support and in solution. These compounds were conjugated to 5 nm and 10 nm gold nanoparticles. These amine and guanidinium functionalized gold nanoparticles were characterized by UV spectroscopy and electron microscopy and found to be stable for about 12 hours. The amine functionalized gold colloid was more stable than the guanidinium functionalized gold colloid. These functionalized gold colloids were found to adhere to the surface of cells (HepG2 and HeLa) and were taken up by endocytosis. No nuclear localization of these gold conjugates was observed.

Degree

Ph.D.

Advisors

Bergstrom, Purdue University.

Subject Area

Pharmacology|Immunology|Organic chemistry

Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server
.

Share

COinS