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Reports Results and discussion


To demonstrate the activity of a self- cleaving deoxyribozyme, we sought to prepare a collection of ssDNAs of defined sequence and length, wherein RCA ampli- fication products ranged from a single- unit DNA (100 nucleotides) to greater than 10 unit DNA repeats in the concate- meric sequence (Figure 2). We reasoned that such a range of DNA products might be useful as ssDNA size markers (DNA ladders) for gel electrophoresis applications. Our studies were initiated by preparing a 100-nucleotide circular DNA template for RCA. Tis was generated from a synthetic DNA template prepared by solid-phase chemical synthesis. Te synthetic DNA template includes a 44-nucleotide sequence complementary to deoxyribozyme I-R3 along with 56 randomly-chosen nucle- otides. Te synthetic DNA was ligated to form a ssDNA circle by using CircLigase, a protein enzyme that efficiently couples a linear DNA carrying both 5′ phosphate and 3′ hydroxyl termini (Figure 2, i) (18). 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-hydro- lyzing deoxyribozyme. However, the deoxy- ribozyme does not cleave until it is exposed to the conditions needed for robust self-pro- cessing (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. Tis 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. Te distribution of products generated


by implementing our RCA/self-cleaving deoxyribozyme scheme was examined by denaturing (8 M urea) 8% PAGE (Figure 3). Te 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 elimi- nated when the circular ssDNA template concentration is reduced from 200 to 40 nM during the RCA amplification (Figure 3b). Importantly, this reduction in template


Vol. 54 | No. 6 | 2013 a M


0 2 5 15 30 1 2 min


h nts


400 500 600 700 800 900 1000


300 200 b


2 0 12 hh


M nts


400 500 600 700 800 900 1000


300 200


C-100 C-100 100 5X template 5X 1X


Figure 3. Gel electrophoresis of the 100mer ssDNA ladder. (a) PAGE separation of the products of rolling circle amplification (RCA) subjected to incomplete deoxyribozyme self-cleavage. M designates a marker lane containing a mixture of linear and circular 100mer template ssDNAs. C-100 designates the circular 100mer DNA template. Asterisks identify bands possibly corresponding to circular template DNAs in- teracting with RCA products. Bands were visualized by staining with SYBR Gold. (b) PAGE separation of RCA and deoxyribozyme cleavage products generated using 40 nM instead of 200 nM of the template DNA concentration used in (a). Annotations are as described in (a).


concentration does not compromise the yield of RCA products. Tus, 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 suffi- cient to yield near equal intensities for the ten bands ranging in size from 100 to 1000 nucleotides.


340 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 available


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