Cleaving DNA with DNA

Abstract

A DNA structure is described that can cleave single-stranded DNA oligonucleotides in the presence of ionic copper. This "deoxyribozyme" can self-cleave or can operate as a bimolecular complex that simultaneously makes use of duplex and triplex interactions to bind and cleave separate DNA substrates. Bimolecular deoxyribozyme-mediated strand scission proceeds with a kobs of 0.2 min-1, whereas the corresponding uncatalyzed reaction could not be detected. The duplex and triplex recognition domains can be altered, making possible the targeted cleavage of single-stranded DNAs with different nucleotide sequences. Several small synthetic DNAs were made to function as simple "restriction enzymes" for the site-specific cleavage of single-stranded DNA.

Figure 1

Sequence and predicted secondary structures of minimized self-cleaving DNAs. (A) Sequence and secondary structure of a synthetic 69-nucleotide self-cleaving DNA that was isolated by in vitro selection. Numbers identify nucleotides that correspond to the 50-nucleotide random-sequence domain that was included in the original DNA pool (note that 19 bases of this domain have been deleted). The conserved nucleotides (nucleotides 11–31, boxed) are similar to those previously used to define this class of deoxyribozymes (16). (B) A 46-nucleotide truncated version of class II DNAs that retains full activity. I and II designate stem–loop structures of the 46-mer that are predicted by the structural folding program dna mfold (18, 19) and that were confirmed by subsequent mutational analysis (Fig. 2). The conserved core of the deoxyribozyme spans nucleotides 27–46 and the major site of DNA cleavage is designated by the arrowhead. Encircled nucleotides can be removed to create a bimolecular complex where nucleotides 1–18 constitute the substrate subdomain, and nucleotides 22–46 constitute the catalyst subdomain.

Figure 2

Confirmation of stems I and II by mutational analysis. (A) Trace amounts of 5′-³²P-labeled substrate DNAs (s1–s3) were incubated with 5 μM complementary or noncomplementary catalyst DNAs (c1–c3) in reaction buffer A containing 10 μM CuCl₂ at 23°C for 15 min. Reaction products were separated by denaturing PAGE on 20% gels and imaged by autoradiography. Bracket identifies the position of the substrate cleavage products. (B) Self-cleavage activity of the original 46-mer sequence compared with the activity of variant DNAs with base substitutions in stem II. Individual 46-mer variants (100 pM 5′-³²P-labeled precursor DNA) were incubated for the times indicated under reaction conditions as described above. Clv1 and Clv2 identify 5′ cleavage fragments produced upon precursor DNA (Pre) scission at the primary and secondary sites, respectively. Mutated positions use the numbering system given in Fig. 1B.

Figure 3

Identification of a triplex interaction between substrate and catalyst DNAs. A revised structural representation portrays a triple-helix interaction (dots) between the four base pairs of stem II and four consecutive pyrimidine residues near the 5′ end of the substrate DNA. c4 and s4 represent sequence variants of c3 and s3 that retain base pairing within stem II and that use an alternate sequence of base triples. DNA cleavage assays were conducted as described in Fig. 2A. Bracket identifies the position of the substrate cleavage products.

Figure 4

Targeted cleavage of DNA substrates with deoxyribozymes containing engineered duplex and triplex recognition elements. (A) A 101-nucleotide DNA incorporating three different deoxyribozyme cleavage sites was prepared by automated chemical synthesis. Each cleavage site consists of an identical leader sequence (shaded boxes) followed by a stem I recognition element of unique sequence. The specific base complementation between the synthetic catalyst DNAs c1, c3, and c7 is also depicted. The catalytic core sequences and the leader sequence–stem II interactions for each site are identical (Inset). Large dots indicate G⋅T wobble pairs that allow cross-reaction between c1 and the target for c3. Small dots indicate base triple interactions. (B) Cleavage of the 101-mer DNA by c1, c3, and c7 was examined by incubating trace amounts of 5′-³²P-labeled substrate in reaction buffer containing 30 μM CuCl₂ at 23°C for 20 min in the absence (−) or presence of 5 μM catalyst DNA, as indicated. Reaction products were separated by denaturing PAGE on 10% gels and visualized by autoradiography. (C) Similarly, a 100-nucleotide DNA was prepared that contained three identical stem I pairing regions (shaded boxes) preceded by eight successive pyrimidine nucleotides of unique sequence. Three synthetic deoxyribozymes (c9, c10, and c11) that carry identical stem I pairing elements (Inset) and extended stem II subdomains of unique sequence were designed to target the three cleavage sites exclusively through DNA triplex interactions. (D) Cleavage of 100-mer DNA by c9, c10, and c11 was established as described in B above. Miscleavage is detected for each triplex-guided deoxyribozyme upon extended exposure during autoradiography (e.g., c11), indicating that weak-forming triplex interactions allow some DNA cleavage activity to occur.

Figure 5

Cleaving double-stranded DNA. Trace amounts of single-stranded (ss) or double-stranded (ds) 101-mer DNA (target strand is 5′-³²P-labeled) was incubated in buffer A containing 10 μM CuCl₂ either without (−) or with (+) 5 μM c3. Single-stranded DNA and the first lane of double-stranded DNA was incubated at room temperature without thermocycling for 15 min. The remainder of the double-stranded DNA samples were incubated in the presence of 5 μM c3 either without (0) or with thermal denaturation (1–12 cycles) as indicated. Thermocycling consisted of repetitive heating and cooling between 94°C (1 min) and 25°C (15 min). Numbers indicate the iterations of thermal cycling and Clv identifies the DNA cleavage product. Reaction products were separated by denaturing PAGE in 10% gels and visualized by autoradiography.