Date of Award

1993

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Plant, Environmental Management and Soil Sciences

First Advisor

Doo-Youn Cho

Abstract

We proposed that known helix-stabilizing, minor-groove binding agents such as CC-1065 and its analog, U-71,184, might be feasibly targeted to specific mRNA by tethering them to antisense oligonucleotides. The CC-1065-tethered antisense oligonucleotide might be hyperstabilized to the target mRNA and arrest the translation of mRNA. Our hypothesis is that these agents can bind and increase the stability of RNA-DNA duplexes which may occur between mRNA and antisense oligonucleotides. To verify our hypothesis, we evaluated the ability of CC-1065, U-71,184, and distamycin A to bind and hyperstabilize the 20-mer sense sequence (5$\sp\prime$-TTACTTCAGTTATGAGACCA-3$\sp\prime)$ duplexed with complementary (antisense) oligonucleotide sequence made with phosphodiester (PO), phosphorothioate (PS), or methyl phosphonate (MP) linkages. Sense DNA sequences (20 mer), when duplexed with PO, PS, or MP DNA oligonucleotides, were saturated at 2, 2, and 1 CC-1065 molecules per duplex, respectively, or at 1 molecule of U-71,184 for each duplex. Each molecule of CC-1065 and U-71,184 was able to bind to poly(rA)-oligo(dT)$\sb{20}$ at maximum binding. Similarly, a sense RNA sequence duplexed with either PO or PS structures was saturated at one CC-1065 molecule per duplex. It was apparent from the melting temperature study that CC-1065 was generally more effective than U-71,184 against DNA-DNA duplexes even if the antisense DNA strand has modified backbone structures. Only CC-1065 was able to hyperstabilize the RNA-DNA duplex as evidenced by a 29$\sp\circ$C increase of the melting temperature. U-71,184 and distamycin A were able to increase the melting temperature of RNA-DNA duplex only about 2$\sp\circ$C and 4$\sp\circ$C, respectively. Translation (wheat germ extract) was inhibited in a dose-dependent manner at antisense:mRNA ratios ranging from 10 to 72. Concomitant incubations of the duplexes with CC-1065 caused significantly enhanced depression of translation at the higher doses. CC-1065 appears to induce hyperstabilization between the antisense oligonucleotide and the mRNA target and arrest translation. Further development of hyperstabilizing antisense oligonucleotide structures is amply justified.

Pages

138

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