Gregory W. Muth, Ph.D. October, 1999                                                                                                Chemistry

I.  Synthesis, bio-conjugation, electronic and spectral properties of phenanthroline analogs
II.  Phenanthroline cleavage of RNA

Research Advisor: Charles M. Thompson, Ph.D.

 ABSTRACT

 In an effort to elucidate the complexities of ribosomal RNA structure, a series of novel phenanthroline-copper complexes were employed to act as chemical nucleases.  It is with these compounds that information regarding the efficiency and specificity of cleavage of RNA by phenanthroline-copper complexes has been collected.  Two separate positional substitutions on the 1,10-phenanthroline template were made; the first at the C-2 carbon yielding N-acetyl-2-aminomethyl-1,10-phenanthroline and N-iodoacetyl-2-aminomethyl- 1,10-phenanthroline.  The second substitutions at the C-5 carbon yielded 5-acetamido-1,10- phenanthroline,  5-( -bromohexanoamido)-1,10-phenanthroline, N-[5-1,10-phenanthrolyl0]-  -bromo-p-toluamide.  Radiolabeled tags were prepared of each positional isomer to study the purification of the bio-conjugated prior to targeted scission. The ability of each substituted phenanthroline to bind Cu(II) was measured spectrophotometrically.  The copper complexes of 2,9-dimethyl-1,10-phenanthroline, C-2 substituted and unsubstituted phenanthroline  were prepared and the cleavage efficiency of each complex compared, with the 1:1 oP-Cu complex of C-5 or unsubstituted phenanthroline showing the most robust scission of RNA, particularly at the postulated pseudoknot  in the 100 region of 23S rRNA.

 Phenanthroline was used as a targeted chemical nuclease, to help elucidate the regions of rRNA which are near-neighbors of the stem-loop structure centering at nucleotide 790 in the 16S rRNA of the E. coli 30S ribosomal subunit.  Using phenanthroline covalently attached to a DNA oligomer complementary to nucleotides 787-795, nucleotides 582-584, 693-694, 787-790 and 795-797 were cleaved robustly and must lie within about 15Å of  the tethered site at the 5'-end of the DNA oligomer, which is adjacent to nucleotide 795 of 16S rRNA.

 Using phenanthroline-copper, covalently attached to a DNA oligomer complementary to nucleotides in the decoding region (1396-1403) it was determined that nucleotides 923- 929, 1391-1396 and 1190-1192 are within approximately 15  of an adjacent position to nucleotide 1403 in inactive subunits.  Additionally, cleavage of these proximal sites is removed or greatly attenuated upon activation of the subunits, while in the active state only cleavages (1404-1405) immediately proximal to the 5' end of the hybridized  probe are present.  These results suggest dynamic interactions within the 30S ribosomal subunit which may allow the discrete structural changes that allow the binding of tRNA in active subunits to be mapped.
 Further studies of targeted scission using the phenanthroline-DNA probe showed an increase in cleavage efficiency in the presence of exogenous hydrogen peroxide.  These conditions provided evidence that the oP-Cu cleavage reaction can be tuned to produce a mild or robust cleavage reaction, a unique property never demonstrated with an organo- metallic chemical nuclease.

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