Precision sequence-specific cleavage of nucleic acids by minor-groove directed metal binding ligands linked through N-2 of deoxyguanosine

Norman Phillip Gerry, Purdue University

Abstract

1,10-Phenanthroline was covalently linked from its 2-position to the N$\sp2$ of deoxyguanosine through two tethers, -$\rm OCH\sb2CH\sb2CH\sb2$- or -$\rm(O)CNHCH\sb2CH\sb2$-, to give N$\sp2$- (3-(phenanthrolin-2-yloxy)propyl) -2$ \sp\prime$-deoxyguanosine, G*, and N$\sp2$- (2-(phenanthrolin-2-yl-carboxamido)ethyl) -2$ \sp\prime$-deoxyguanosine, G$\sp{\wedge},$ (Figure 1). These nucleosides were incorporated into oligonucleotides bearing the sequence 5$\sp\prime$-TCTCAGTGGTATTTG-3$\sp\prime$ using standard phosphoramidite chemistry. A single deoxyguanosine was replaced by G* or G$\sp{\wedge}$ in each sequence synthesized. In addition, two disubstituted oligonucleotides were prepared, substituting G* or G$\sp{\wedge}$ at positions 6 and 9. Two 31-mers, 5$\sp\prime$-CTGGCTCACAAATACCA CTGAGATCT TTT TC-3$\sp\prime,$ containing the complementary sequence were prepared for use as cleavage targets. One 31-mer contained a biotinylated thymidine at position 30 for detection by chemiluminescence while the other was 5$\sp\prime$-$\sp{33}$P labeled for detection by autoradiography and phosphor imaging. In the presence of Fe(II), oxygen, and dithiothreitol (25$\sp\circ$C, pH 7.4, 5 hours), the G* and G$\sp{\wedge}$ containing oligonucleotides afforded site-specific cleavage of the complementary sequence. Results of cleavage experiments showed that when the phenanthroline was located at the end of the oligonucleotide, cleavage occurred over a 10 nucleotide region. In contrast, sequences in which the phenanthroline was tethered near the middle of the oligonucleotide led to highly specific cleavage concentrated at one or two sites. The precision and specificity for cleavage at a single site appeared to be greater for the G$\sp{\wedge}$ containing oligonucleotides. This is a noteworthy result because the amide linked phenanthroline has less conformational freedom than the ether linked phenanthroline. In addition, modeling studies showed the amide bound phenanthroline to be positioned more deeply in the minor groove than the ether bound phenanthroline.* ftn*Please refer to the dissertation for diagram.

Degree

Ph.D.

Advisors

Bergstrom, Purdue University.

Subject Area

Organic chemistry|Biochemistry

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