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ab P2 C C


A A A G


T T


G A T


P1 A T


5'


GC C A


G


G T


T A


GC A I-R3 A G C T G T C G T T G O


-O P O


HO- H


5′ O O O A


Zn2+ I-R3


O O P


HO O O O


Figure 1. Schematic representation of a class I self-hydrolyzing deoxyribozyme and its chemical reac- tion. (a) Sequence and secondary structure of the I-R3 self-cleaving deoxyribozyme. Nucleotides in red are highly-conserved and the arrowhead identifies the site of hydrolysis. (b) Proposed reaction for hydrolysis of the 3’ phosphoester linkage to yield products with 3’ hydroxyl and 5’ phosphate termini.


- A OH


O and samples were used for electropho- resis mobility assays using agarose gel.


Purification of ss100 DNA ladder products Te ten shortest ss100 DNA ladder products (ranging from 100 to 1000 nucleotides) were separated by denaturing (8 M urea) 8% PAGE. Te bands were visualized by UV shadowing, individually excised from the gel, and combined in one tube prior to elution by crush-soaking overnight in 10 mM Tris-HCl (pH 7.5 at 23°C), 200 mM NaCl, and 1 mM EDTA. DNA was recovered from solution by the addition of 2.5 volumes 100% ethanol followed by centrifugation. Te resulting DNA pellet was resuspended in deionized H2


5' p synthetic linear ssDNA


CircLigase i


circular ssDNA template 5'


I-R3 5' concatemeric ssDNA product 3X I-R3 I-R3


ii (primer) DNA Pol


>10X


7X 6X


5X 4X


10X 9X 8X


Oligonucleotides A list of oligonucleotides used in this study is provided below. Underlined sequences in the template strands encode the I-R3 deoxy- ribozyme. ss50, ss100, and ss200 refer to the template DNAs used to generate ssDNA ladders of the increment lengths indicated.


ss50 DNA Template 5′-pTAGGTAACGCTTCAACGT- CACATTCTGTGACAGCTCAAC- TACGTTACTTG


ss50 DNA Primer 5′-GTTACCTACAAGTAACGTA


ss100 DNA Template 5′-pCTTGACTGCTTATGAGCAT- GGTGTATATGTGCCGAATTAGG- TAACGCTTCAACGTCACATTCT- GTGACAGCTCAACTACGTTACT- TGGTCTGCAATGATA


1X 5' 2X iii Zn2+


partial deoxyribozyme digest products Clv


Clv


separation iv


2X


ss100 DNA Primer 5′-AGCGTTACCTAATTCG


1X


Figure 2. Scheme for producing an ssDNA ladder using RCA and a self-hydrolyzing deoxyribozyme. Synthetic linear DNAs carrying a complement of a self-hydrolyzing deoxyribozyme are (i) treated with CircLigase to create circular ssDNAs. The resulting DNA templates are (ii) incubated with DNA primer and Phi 29 DNA polymerase to generate concatemeric ssDNA products. These products are (iii) incubated in the presence of Zn2+


to promote cleavage of the deoxyribozymes. Partial digest of


the deoxyribozymes (~single-hit kinetics) yields a variety of ssDNA lengths that differ in size by the unit-length (1X) of the template DNA, which is evident upon (iv) separation of the products by gel electrophoresis.


Vol. 54 | No. 6 | 2013 338


ss200 DNA Template 5′-pCTTGACTGCTTATGAGCAT- GGTGTATATGTGCCGAATTAGG- TAACGCTTCAACGTCACATTCT- GTGACAGCTCAACTACGTTACT- TGGTCTGCAATGATAGAATGTG- GTATTCCTAAATCTCAACTGAT- GAATCTTTCTACCTGTAATAAT- GTTGTTCCGTTAGTTCGTAT- GATTAACGTAGATATCTCTCCT- CAGCATA


ss200 DNA Primer 5′-AGCGTTACCTAATTCG


www.BioTechniques.com A


G A T


G T G A O O 3′ 2′ A O O A


PAGE or 1.5% agarose electrophoresis under denaturing or non-denaturing conditions as indicated for each experiment. Bands were visualized with SYBR Gold nucleic acid gel stain (Invitrogen, Grand Island, NY, USA) and imaged by UV transillumination.


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