Upon incubation of this circular DNA template with Phi 29 DNA polymerase and a complementary DNA primer, a single-stranded concatemer consisting of multiple linear copies of the sequence complementary to the circular template is produced (Figure 2, ii). At the junction of each DNA repeat resides the sequence corresponding to the I-R3 class I self-hydrolyzing deoxyribozyme. However, the deoxyribozyme does not cleave until it is exposed to the conditions needed for robust self-processing (50 mM HEPES, pH 7.0 at 23°C; 100 mM NaCl; 2 mM ZnCl2) (Figure 2, iii). By halting the reaction before all of the deoxyribozymes have cleaved, a mixture of products representing a range of unit-length DNAs is generated. This mixture of deoxyribozyme cleavage products can serve as an ssDNA ladder when separated by gel electrophoresis (Figure 2, iv) or by other methods that can separate large ssDNAs.
The distribution of products generated by implementing our RCA/self-cleaving deoxyribozyme scheme was examined by denaturing (8 M urea) 8% PAGE (Figure 3). The smallest of the ten major bands of the sample, which span from 100 to 1000 nucleotides, are well resolved, whereas larger product bands are less well resolved (Figure 3a). Initially, we also observed a few unanticipated bands (Figure 3a, asterisks), perhaps caused by the presence of excess circular DNA template interacting with amplification products; these are eliminated when the circular ssDNA template concentration is reduced from 200 to 40 nM during the RCA amplification (Figure 3b). Importantly, this reduction in template concentration does not compromise the yield of RCA products. Thus, the RCA reaction followed by a short incubation under deoxyribozyme cleavage conditions permit the generation of an ssDNA ladder of 100-nucleotide increments (termed ss100 DNA ladder) that is free of undesired bands (Figure 3b). It should be noted our system can permit biases toward production of short or long concatemers, simply by increasing or decreasing catalysis time, respectively. In our hands, an incubation time of approximately 30 min was sufficient to yield near equal intensities for the ten bands ranging in size from 100 to 1000 nucleotides.
When seeking size markers for ssDNAs, some researchers use denaturing conditions to create ssDNAs from double-stranded DNAs (dsDNAs) of known length. However, incomplete denaturation can cause confusion since ssDNA and dsDNA have different electrophoretic mobilities. We and others have occasionally resorted to using single-stranded RNAs (ssRNAs) as surrogates for ssDNA size markers, but again, the difference in mobility between DNA and RNA can cause confusion.
To illustrate this latter effect, we compared the electrophoretic mobilities of the ss100 DNA ladder constituents with ssRNAs in the commercially availableRNA marker preparation RiboRuler (Fermentas Inc., Lafayette, CO, USA). Electrophoretic separation of the ss100 DNA ladder and RiboRuler nucleic acids in adjacent lanes of either denaturing or non-denaturing agarose gels revealed substantial differences in the mobilities of bands (Figure 4a). For example, the DNAs in the ss100 DNA ladder generated consistent banding patterns, whereas the RNAs in the RiboRuler sample exhibit some differences between denaturing and non-denaturing conditions, likely due to the formation of strong RNA structures by particular RNA sequences. Also, the DNA and RNA molecules of equal size do not co-migrate, which highlights the disadvantages of using RNA markers as surrogates for ssDNA. Likewise, differences in gel mobility between these DNAs and RNAs are also observed when the two samples are separated by denaturing (Figure 4b) and non-denaturing (Figure 4c) PAGE.
The method used to produce the ss100 DNA ladder can be used to generate markers of any unit size increment simply by varying the number of nucleotides in the template DNA. For example, the addition of six nucleotides to the 44 nucleotides of the I-R3 deoxyribozyme complementary sequence yielded 50-nucleotide unit-length ssDNA products (ss50 DNA ladder) (Figure 4d) whereas the addition of 156 nucleotides yielded ssDNA markers with 200-nucleotide increments (ss200 DNA ladder) (Figure 4e).
Thus, our combined RCA/self-cleaving deoxyribozyme scheme allows for the production of ssDNA markers with increments of ~50 nucleotides or larger. Furthermore, ssDNA markers produced by this method can be easily internally- or 5’-radiolabeled using standard methods. For example, radiolabeling using γ-32P[ATP] and polynucleotide kinase can be carried out after removal of the 5’ phosphate group generated by deoxyribozyme hydrolysis. This makes possible the production of ssDNAs for use as markers that can overcome the problems of structure formation and altered mobility observed with some existing RNA markers. Moreover, since DNA is more stable than RNA, ssDNA markers will have a storage time that is far greater than thatof RNA markers. Samples of the ss100 DNA ladder can be obtained for evaluation or application from the Coli Genetics Stock Center at Yale University (http://cgsc.biology.yale.edu/).
In summary, we have developed a simple and effective method to produce ssDNAs of defined sequence and length from engineered circular DNA templates. This approach permits the efficient synthesis of DNAs that can be much longer and carry less chemical damage than those prepared by existing solid-phase DNA synthesis methods. In the current study, we demonstrate the use of such ssDNA products as markers for gel electrophoresis applications. Markers of this type could be useful when conducting experiments on natural ssDNAs (e.g., bacteriophage genomes) or on cDNA products made from natural RNAs. Additional applications involving complete digestion with a deoxyribozyme should permit the production of uniform-length sequence specific ssDNAs for other uses. Moreover, one could envision the incorporation of deoxyribozymes or DNA aptamers with other functions that would yield multifunctional DNA constructs produced by RCA.
We thank the Breaker laboratory for helpful discussions. This work was supported by grants from DARPA and the NIH (GM022778). Research in the Breaker laboratory is also supported by the Howard Hughes Medical Institute.
The authors have filed for intellectual property protection on aspects of this work.
Address correspondence to Ronald R Breaker, Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA. Email: firstname.lastname@example.org
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