A new class of genome rare cutters.

Although significant efforts have been directed at developing efficient techniques for rare and super rare genome cutting, only limited success has been achieved. Here we propose a new approach to solve this problem. We demonstrate that peptide nucleic acid 'clamps' (bis-PNAs) bind strongly and sequence specifically to short homopyrimidine sites on lambda and yeast genomic DNAs. Such binding efficiently shields methylation/restriction sites which overlap with the bis-PNA binding sites from enzymatic methylation. After removing the bis-PNA, the genomic DNAs are quantitatively cleaved by restriction enzymes into a limited number of pieces of lengths from several hundred kbp to several Mbp. By combining various bis-PNAs with different methylation/restriction enzyme pairs, a huge new class of genome rare cutters can be created. These cutters cover the range of recognition specificities where very few, if any, cutters are now available.

Protonated pyrimidine-purine-purine triplex.

We have studied a protonated pyrimidine-purine-purine (Py-Pu-Pu) triplex, which is formed between the d(C)nd(G)n duplex and the d(AG)m oligonucleotide as the third strand and carries the CG*A+ protonated base-triads. We have observed such an intermolecular complex between a plasmid carrying the d(C)18 d(G)18 insert and the d(AG)5 oligonucleotide without bivalent cations in 200 mM of Na+ at pH4.0. Bivalent cations additionally stabilize the complex. We propose the structures for nearly isomorphous base-triads TA*A, CG*G and CG*A+. To identify the H-DNA-like structure, which includes the triplex between d(C)n d(G)n duplex and the AG-strand, we have cloned in a superhelical plasmid the insert: G10TTAA(AG)5. The data on photofootprinting and chemical modification with diethyl pyrocarbonate, potassium permanganate and dimethyl sulfate demonstrate that the H-like structure with triplex carrying CG*G and CG*A+ base triads is actually formed under acid conditions. In the course of this study we have come across unexpected results on probing of Py-Pu-Pu triplexes by dimethyl sulfate (DMS): the protection effect is observed not only for guanines entering the duplex but also for guanines in the third strand lying in the major groove. We have demonstrated this effect not only for the case the novel protonated Py-Pu-Pu triplex but also for the traditional non-protonated Py-Pu-Pu intramolecular triplex (H*-DNA) formed by the d(C)37 d(G)37 insert in supercoiled plasmid in the presence of Mg2+ ions.

Kinetic trapping of H-DNA by oligonucleotide binding.

Homopurine-homopyrimidine mirror repeats are known to adopt the H form under acidic pH and/or negative supercoiling. In H-DNA, one half of the purine strand enters the triplex whereas the second half is unstructured and can form duplex with complementary oligonucleotide. However, because the same oligonucleotide can form triplex with the homopurine-homopyrimidine insert, one could expect that oligonucleotide would make H-DNA thermodynamically less favorable, as was claimed by Lyamichev et al. Nucl. Acids Res. 16, 2165-2178 (1988). Now we show that complex between oligonucleotide and H-DNA, formed under conditions favorable for the H-form extrusion, is kinetically trapped in superhelical DNA and remains stable up much higher pH values than H-DNA alone. Experiments on chemical probing show that such complex exists for a plasmid with native superhelical density at pH7. We have also used this approach to demonstrate a pH-dependent structural transition in yeast telomeric sequence, d(CACACCCA)16.

An eclectic DNA structure adopted by human telomeric sequence under superhelical stress and low pH.

We have found, with the aid of 2-D gel electrophoresis, that double-stranded human telomeric repeat, (T2AG3)12.(C3TA2)12, being cloned within a plasmid, forms a protonated superhelically-induced structure. Experiments on chemical and enzymatic probing also indicate that the human telomeric repeats adopt an unusual structure. We have proposed an eclectic model for this structure in which four different elements coexist: a non-orthodox intramolecular triplex stabilized by the canonical protonated C.G*C+ base-triads and highly enriched by noncanonical base-triads; the intramolecular quadruplex formed by a portion of the G-rich strand; the single-stranded region encompassing a portion of the G-rich strand and, probably, the (C,A)-hairpin formed by a portion of the C-rich strand.

Formation of intramolecular triplex in homopurine-homopyrimidine mirror repeats with point substitutions.

We have used two-dimensional gel electrophoresis to study the structural transition to the triplex H form of sequences 5'-AAGGGAGAAXGGGGTATAGGGGYAAGAGGGAA-3' where X and Y are any DNA bases. The transition was observed at acid pH under superhelical stress. For X = Y = A or X = Y = G the sequences corresponded to homopurine-homopyrimidine mirror repeats (H-palindrome) which are known to adopt the H form under acid pH and superhelical stress. We have shown that the H form is actually formed for all X and Y, though in cases other than X = Y = A and X = Y = G the transition requires larger negative superhelical stress. Different substitutions require different superhelicity levels for the transition to occur. Theoretical analysis of the data obtained made it possible to estimate the energy cost of triplex formation due to all possible mismatched base triads.