Reports Reports
Production of single-stranded DNAs by self-cleavage of rolling-circle amplification products Hongzhou Gu1,2
and Ronald R. Breaker1,2,3
1Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, United States, 2 Connecticut, United States, and 3
BioTechniques 54:337-343 (June 2013) doi 10.2144/000114009 Keywords: deoxyribozymes; self-hydrolysis; rolling-circle amplification; single-stranded DNA ladder
DNA molecules that encode a small, high-speed self-hydrolyzing deoxyribozyme are used as templates for rolling circle amplification (RCA) to produce single-stranded DNAs (ssDNAs) of single- and multiple-unit lengths. Including self- cleaving deoxyribozymes in RCA products can generate large amounts of ssDNAs with defined sequence and length as well as precise termini. We also demonstrate the use of this method to efficiently generate ssDNA size markers by using deoxyribozyme reaction conditions that permit partial processing.
Deoxyribozymes are DNA molecules that form structures capable of catalyzing chemical reactions (1–3). Given the central role of DNA in genetic information storage and its impor- tance in biotechnology, deoxyribozymes might find utility in engineered organisms or as reagents for various molecular applications (4–6). Of particular interest to us are DNAs that catalyze self-processing reactions (7–10). Such deoxyribozymes could be harnessed to create DNA constructs that become modified based on their inherent catalytic activities when exposed to specific reaction conditions. For example, engineered self-cleaving deoxy- ribozymes that employ oxidation (7), depuri- nation (8), or hydrolysis (9–12) mechanisms have been created by using various directed evolution strategies. Self-cleaving deoxy- ribozymes that operate with appropriate reaction rates and chemical characteristics might find broad utility for various applica- tions involving DNA cleavage. Recently, we identified two classes of
engineered self-cleaving deoxyribozymes that hydrolyze DNA with high speed and sequence specificity (13). One such deoxy- ribozyme, named I-R3 (Figure 1a), carries a small catalytic core composed of 17 nucle- otides flanked by either 1 or 2 base-paired substructures. Representatives of this deoxy- ribozyme class exhibit an observed rate
constant (kobs) for DNA hydrolysis of ~1 min-1
(half-life of ~40 s) when incubated at near neutral pH and in the presence of millimolar concentrations of Zn2+
. Tis deoxyribozyme
cleaves the phosphoester bond between the 3′ oxygen and the phosphorus center of an ApA linkage to yield a 3′ cleavage fragment with a 5′ phosphate group (Figure 1b). We speculated that an efficient self-
cleaving deoxyribozyme would be useful for cleaving multimeric ssDNA products that are generated by RCA. RCA generates concatemer DNA products since DNA polymerase is using a circular DNA template (14,15). Whereas most applications involving RCA exploit its ability to amplify weak biochemical signals in various diagnostic systems (16,17), a self-cleaving deoxyribozyme would permit the concatemers to be resolved into unit-length DNA products.
Materials and methods
Rolling circle amplification (RCA) A 10 µL or 2 µL aliquot of 1 µM circular DNA template prepared by CircLigase (Epicenter Biotechnologies, Madison, WI, USA) according to the manufacturer’s directions was combined with an equivalent molar amount of primer in a total of 50 µL containing 40 mM Tris-HCl (pH 7.5 at 23°C), 50 mM KCl,
mM DTT. To ensure binding of the primer to the template, an annealing procedure was performed by stepwise 2-min incubations at 80°C, 60°C, 45°C, and 23°C. Aſter annealing, 1 µL of 10 mM dNTPs, 1 µL of 10 mg/mL BSA, and 2 µL of 10 unit/µL Phi 29 DNA polymerase (New England BioLabs, Ipswich, MA, USA) were added to initiate DNA synthesis. Te reaction was incubated at 30°C for 4 h and stopped by inactivating the enzyme at 65°C for 10 min. Te products were precipitated with 2.5 volumes of 100% ethanol, centrifuged to recover the DNA pellet, and resuspended in a 50 µL solution containing 50 mM HEPES (pH 7.0 at 23°C) and 100 mM NaCl.
10 mM MgCl2 , 5 mM (NH4)2 SO4
Self-hydrolyzing deoxyribozyme reactions Te ssDNA concatemers from RCA were allowed to fold by 2-min stepwise incubations at 80°C, 60°C, 45°C, and 37°C. At 37°C, the self-cleavage reaction was initiated by mixing the above 50 µL solution with an additional 50 µL containing 50 mM HEPES (pH 7.0 at 23°C), 100 mM NaCl, and 4 mM ZnCl2
.
At different time points (2 min, 5 min, 15 min, 30 min, 1h, and 2h), a 10 µL aliquot was removed and mixed with 10 µL stop buffer containing 95% formamide and 20 mM EDTA. Products were separated by 8%
Method summary: Here we report a novel method to generate large amounts of single-stranded DNA of defined length and sequence using self-hydrolyzing deoxyribozymes.
Vol. 54 | No. 6 | 2013 Vol. 54 | No. 6 | 2013 337
www.BioTechniques.com www.BioTechniques.com , and 4 Howard Hughes Medical Institute, New Haven, Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA
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