Sequence-specific double-strand cleavage of DNA by penta-N-methylpyrrolecarboxamide-EDTA X Fe(II).

Abstract

In the presence of O2 and 5 mM dithiothreitol, penta-N-methylpyrrolecarboxamide-EDTA-Fe(II) [P5E*Fe(lI)] at 0.5 ILM cleaves pBR322 plasmid DNA (50 pAM in base pairs) on opposite strands to afford discrete DNA fragments as analyzed by agarose gel electrophoresis. High-resolution denaturing gel electrophoresis of a nP-end-labeled 517-base-pair restriction fragment containing a major cleavage site reveals that P5E-Fe(li) cleaves 3-5 base pairs contiguous to a 6-base-pair sequence, 5'-T-T-T-T-T-A-3' (4,323-4,328 base pairs). The major binding orientation of the pentapeptide occurs with the amino terminus at the adenine side of this sequence. In the presence of 5 mM dithiothreitol, 0.01 pzM P5E-Fe(II) converts form I pBR322 DNA at 0.22 IzM plasmid (1.0 mM in base pairs) to 40% form II, indicating the cleavage reaction is catalytic, turning over a minimum of nine times. This synthetic molecule achieves double-strand cleavage of DNA (pH 7.9, 25QC) at the 6-base-pair recognition level and may provide an approach to the design of "artificial restriction enzymes." Restriction endonucleases (type II) cleave double-helical DNA on opposite strands at or close to a defined recognition sequence typically 4-6 base pairs in size (1-3). The ability of these enzymes to cleave double-stranded DNA into unique DNA fragments is useful for DNA sequence determinations, chromosome analyses, gene isolation, and recombinant DNA manipulations. We describe here our initial efforts toward the design of synthetic, sequence-specific, double-strand DNA-cleaving molecules. Attachment of EDTA.Fe(II) to a sequence-specific DNA-binding molecule creates a sequence-specific DNAcleaving molecule (4,5). The natural product distamycin A is a tripeptide containing three N-methylpyrrolecarboxamides that binds in the minor groove of double-helical DNA with a strong preference for A+T-rich regions (6-11). The sequence specificity of distamycin binding presumably results from hydrogen bonding between the amide NHs of the antibiotic and the 0-2 of thymine or the N-3 of adenine (12)(13)(14)(15)

Multiple proteins bind to VA RNA genes of adenovirus type 2.

“…Interestingly, a region of enhanced MPE cleavages was observed on each strand approximately 16 bp upstream of the center of Pr2. This pattern of enhanced cleavages mimics that produced by a fixed EDTA-chelated iron located near the DNA phosphodiester backbone which generates a diffusible, nonspecific DNA cleaving species (i.e., the hydroxyl radical) (36). Thus, the observed cleavages suggest that the intercalation site between base pairs 47 and 48 is a preferred MPE binding site and may be indicative of a protein-induced conformational change in the DNA (such as kinking) which promotes the stability of the MPE-DNA interaction (21).…”

Antiparallel side-by-side dimeric motif for sequence-specific recognition in the minor groove of DNA by the designed peptide 1-methylimidazole-2-carboxamide netropsin.

“…Although there has been some encouraging success with regard to the design and synthesis ofpeptide analogs that bind larger sequences of pure A+T-rich double-stranded DNA (10)(11)(12)(13)(14), there has not been corresponding success in the development of well-understood GC recognition for targets with mixed A*T and G*C sequences (15)(16)(17). Attachment of pyridine-2-carboxamide to the amino terminus of the dipeptide bis-N-methylpyrrolecarboxamide affords the tripeptide pyridine-2-carboxamide netropsin (2-PyN), which binds specifically the sequence 5'-TGACT-3' as well as the family of 5'-(A T)5-3' sequences (18,19).…”

Nonenzymatic sequence-specific cleavage of single-stranded DNA.

“…The yield of 5'-DTPA-P-16-mer was greater than 90%, and the final product was readily purified by chromatography on RPC-5. (2). A further control experiment including Fe2+ and dithiothreitol but omitting all 16-mer derivatives was carried out at the same time.…”

“…When the total radioactivity in lanes [2][3][4][5] (Fig. 4) A reagent that prevents a specific single-stranded RNA from participating in protein synthesis in vivo would obviously have many important applications (11).…”

“…In the presence of 02 and dithiothreitol, the Fe2+ EDTA moiety acts as a source of free radicals, which bring about the cleavage of the DNA backbone by attack on 4' carbon atoms. When EDTA is attached to ethidium bromide, which binds strongly to DNA but with relatively low specificity, the DNA backbone can be cut at many points (1); when EDTA is attached to distamycin or synthetic analogues of distamycin, compounds that bind to A+T-rich regions of DNA, cleavage is restricted to points close to the specific binding sites (2).…”