Hongzhou Gu1,2
and Ronald R. Breaker1,2, 3
1Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, United States2Howard Hughes Medical Institute, New Haven, Connecticut, United States3Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA
BioTechniques, Vol. 54, No. 6, June 2013, pp. 337&343
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 (-). Given the central role of DNA in genetic information storage and its importance in biotechnology, deoxyribozymes might find utility in engineered organisms or as reagents for various molecular applications (-). Of particular interest to us are DNAs that catalyze self-processing reactions (-). 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 deoxyribozymes that employ oxidation (), depurination (), or hydrolysis (-) mechanisms have been created by using various directed evolution strategies. Self-cleaving deoxyribozymes that operate with appropriate reaction rates and chemical characteristics might find broad utility for various applications involving DNA cleavage.
Recently, we identified two classes of engineered self-cleaving deoxyribozymes that hydrolyze DNA with high speed and sequence specificity (). One such deoxyribozyme, named I-R3 (Figure 1a), carries a small catalytic core composed of 17 nucleotides flanked by either 1 or 2 base-paired substructures. Representatives of this deoxyribozyme 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+. This 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 (, ). Whereas most applications involving RCA exploit its ability to amplify weak biochemical signals in various diagnostic systems (, ), 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 &L or 2 &L aliquot of 1 &M 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 &L containing 40 mM Tris-HCl (pH 7.5 at 23&C), 50 mM KCl, 10 mM MgCl2, 5 mM (NH4)2SO4, and 4 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. After annealing, 1 &L of 10 mM dNTPs, 1 &L of 10 mg/mL BSA, and 2 &L of 10 unit/&L Phi 29 DNA polymerase (New England BioLabs, Ipswich, MA, USA) were added to initiate DNA synthesis. The reaction was incubated at 30&C for 4 h and stopped by inactivating the enzyme at 65&C for 10 min. The products were precipitated with 2.5 volumes of 100% ethanol, centrifuged to recover the DNA pellet, and resuspended in a 50 &L solution containing 50 mM HEPES (pH 7.0 at 23&C) and 100 mM NaCl. Method summaryHere we report a novel method to generate large amounts of single-stranded DNA of defined length and sequence using self-hydrolyzing deoxyribozymes.
Self-hydrolyzing deoxyribozyme reactionsThe 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 &L solution with an additional 50 &L 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 &L aliquot was removed and mixed with 10 &L stop buffer containing 95% formamide and 20 mM EDTA. Products were separated by 8%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.
Purification of ss100 DNA ladder productsThe ten shortest ss100 DNA ladder products (ranging from 100 to 1000 nucleotides) were separated by denaturing (8 M urea) 8% PAGE. The 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. The resulting DNA pellet was resuspended in deionized H2O and samples were used for electrophoresis mobility assays using agarose gel. OligonucleotidesA list of oligonucleotides used in this study is provided below. Underlined sequences in the template strands encode the I-R3 deoxyribozyme. ss50, ss100, and ss200 refer to the template DNAs used to generate ssDNA ladders of the increment lengths indicated.ss50 DNA Template5′-pTAGGTAACGCTTCAACGTCACATTCTGTGACAGCTCAACTACGTTACTTGss50 DNA Primer5′-GTTACCTACAAGTAACGTAss100 DNA Template5′-pCTTGACTGCTTATGAGCATGGTGTATATGTGCCGAATTAGGTAACGCTTCAACGTCACATTCTGTGACAGCTCAACTACGTTACTTGGTCTGCAATGATAss100 DNA Primer5′-AGCGTTACCTAATTCGss200 DNA Template5′-pCTTGACTGCTTATGAGCATGGTGTATATGTGCCGAATTAGGTAACGCTTCAACGTCACATTCTGTGACAGCTCAACTACGTTACTTGGTCTGCAATGATAGAATGTGGTATTCCTAAATCTAACTGATGAATCTTTCTACCTGTAATAATGTTGTTCCGTTAGTTCGTATGATTAACGTAGATATCTCTCCTCAGCATAss200 DNA Primer5′-AGCGTTACCTAATTCG Results and discussionTo demonstrate the activity of a self-cleaving deoxyribozyme, we sought to prepare a collection of ssDNAs of defined sequence and length, wherein RCA amplification products ranged from a single-unit DNA (100 nucleotides) to greater than 10 unit DNA repeats in the concatemeric 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. This was generated from a synthetic DNA template prepared by solid-phase chemical synthesis. The synthetic DNA template includes a 44-nucleotide sequence complementary to deoxyribozyme I-R3 along with 56 randomly-chosen nucleotides. The 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) ().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 am 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 ().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.
Acknowledgments
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.
Competing interests
The authors have filed for intellectual property protection on aspects of this
Correspondence
Address correspondence to Ronald R Breaker, Department of Molecular, Cellular
and Developmental Biology, Yale University, New Haven, Connecticut, USA.
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rolling circle amplification: correlation with in vitro assays for serum
Monoclonal antibodiesAllergic reactionAllergyImmunoglobulin EMedical researchMedicine, ExperimentalAllergens
Mullenix, Michael C.Wiltshire, SteveShao, WeipingKitos, GarySchweitzer, Barry
10/01/2001
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Date:&Oct, 2001 Source Volume:&47 Source Issue:&10
Product&Code:&8000200 Medical R 9105220 Health Research P 8000240 Epilepsy & Muscle Disease R&D NAICS&Code:&54171 Research and Development in the Physical, Engineering, and Life
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Full Text:
Allergen-specific IgE antibody in patient serum is used to predict
an allergic response in individuals with concordant clinical history.
For more than 30 years, in vitro assays for allergen-specific IgE have
been used along with or in place of skin-prick allergen testing (SPT)
(1). In vitro test methods include various immunoassay formats with
solid-phase supports such as paper disks, microtiter plates,
nitrocellulose, and microparticles. The field has advanced with
immunoassay refinements, including solid phases with higher
allergen-binding capacities, monoclonal antibodies for detection, enzyme
amplification systems, and fluid-phase allergen/IgE
these have improved the sensitivity of the in vitro assays and provided
better correlation to skin-prick allergen tests (2-4).SPT and in vitro allergen-specific IgE assays have advantages and
disadvantages. SPT results are available immediately, allowing the
allergist to provide treatment while the patient is still in the office.
Multiple allergen extracts are tested simultaneously on the same
patient. Skin-prick tests have the highest positive predictive value
because they are biological, but they also have a high false-positive
rate. To be administered in vivo, the allergen extracts must be sterile
and of low toxicity to prevent anaphylaxis. SPT is expensive because it
requires a skilled practitioner. In vitro serum allergen-specific IgE
immunoassays are semiquantitative and allow testing of an allergic
response over time (4). They are minimally invasive, with no risk of an
adverse reaction in the patient, and are the best option in patients
with severe skin conditions such as eczema, urticaria, or
dermatographism. In food allergy testing, in vitro tests can reduce by
one-half the need for oral allergen challenges (4). In vitro tests can
also be used with patients receiving medications, such as
antihistamines, that may interfere with SPT responsiveness. However,
results of in vitro IgE assays can vary between test formats as well as
between laboratories performing the same assay (5).An adaptation of rolling circle amplification (RCA) (6), termed
"immunoRCA", for very sensitive detection and measurement of
proteins has been described recently (7). In immunoRCA, the 5' end
of an oligonucleotide primer is att thus, in the
presence of circular DNA, DNA polymerase, and nucleotides, the rolling
circle reaction produces a concatamer of circle DNA sequence copies that
remain attached to the antibody. The amplified DNA is detected by
hybridization of fluorescently labeled, complementary oligonucleotide
probes. When performed on a solid phase, immunoRCA allows substantial
multiplexing because signal amplification occurs on the immobilized
detection antibody rather than in solution. ImmunoRCA is thus well
suited as an amplification technique in microarray immunoassays (7).We previously demonstrated that detecting allergen-specific IgE on
microarrays using immunoRCA provided results that were in excellent
agreement those obtained with SPT (8). In the present study, we compare
results obtained by immunoRCA on microarrays with those obtained from
two commercially available allergen-specific IgE assays and one in-house
reference laboratory test. Forty-four serum samples were selected to
provide 6 positive and 5 negative serum samples for each of the
following four allergens: cat dander, dust mites (Dermatophagoides
farinae), short ragweed, and peanuts. The positive serum samples were
prepared by pooling three to five patient sera to provide one positive
serum sample for each of the six concentrations used in the alternative
scoring method (ASM) for allergen-specific IgE assays. We used the
Pharmacia CAP system to assign ASM scores to patients' sera. Each
negative serum sample corresponded to a single patient. All
characterized serum samples were analyzed simultaneously in the
Pharmacia CAP system, the DPC AlaSTAT system, and the Esoterix Allergy
and Asthma system immunoassays to measure correlation to the described
microarray assay. Commercial assay testing was carried out at Esoterix
Allergy and Asthma (Gainesville, FL).ImmunoRCA assays on allergen microarrays were carried out on all 44
samples as described previously with minor modifications (8).
Microarrays of cat dander, dust mite, short ragweed, and peanut extracts
were blocked with 2 g/L bovine serum albumin in 50 mmol/L glycine (pH
9.0) for 1 h at 37[degrees]C. After incubation, the slides containing
the microarrays were washed twice in phosphate-buffered saline (PBS)
containing 0.5 mL/L Tween 20 by soaking for 2 min in Coplin jars.
Patient serum (10 [micro]L) was added to individual microarrays and
incubated for 30 min at 37[degrees]C. The microarrays were washed twice
in PBS containing 0.5 mL/L Tween 20. Biotinylated polyclonal goat
anti-human IgE (20 [micro]L of a 5 mg/L BiosPacific) was added
to each microarray and incubated for 30 min at 37[degrees]C; the
microarrays were then washed twice. An anti-biotin monoclonal antibody
(Jackson ImmunoResearch) conjugated to a 35mer oligonucleotide RCA
primer, as described previously (7), was mixed with 200 nmol/L
complimentary single-stranded circular DNA in PBS containing 0.5 mL/L
Tween 20 and 1 mmol/L EDTA and incubated at 37[degrees]C for 30 min.
Conjugate preannealed to circular DNA (20 [micro]L of a 5 mg/L solution)
was added to each array and incubated at 37[degrees]C for 30 min. After
conjugate binding, the microarrays were washed with PBS containing 0.5
mL/L Tween 20. The RCA reaction was carried out at 37[degrees]C for 30
min with T7 native polymerase, and the product of the RCA reaction was
detected by fluorescently labeled probes as described previously (7).
Slides were scanned on a GSI Lumonics ScanArray 5000 microarray scanner,
and the fluorescence was quantified by QuantArray software.All serum samples that were positive by the Pharmacia CAP assay for
a particular allergen demonstrated a dose-dependent ASM score when IgE
concentrations for that allergen were measured by immunoRCA (Fig. 1).
Furthermore, none of the serum samples negative for a particular
allergen in the Pharmacia CAP assay produced a fluorescence intensity in
the immunoRCA assay that was greater than the corresponding lowest ASM
score sample for that allergen. None of the 44 samples tested gave
false-negative or -positive test results. Although the positive serum
samples were prepared from pools of sera from three to five patients and
may not fully represent an unselected population, the results indicate
that the immunoRCA microarray assay correlates with ASM scores assigned
using the Pharmacia CAP assay.To determine the correlation of the immunoRCA microarray assay with
other commercial assays, the positive and negative serum samples were
tested simultaneously in three commercial allergen-specific IgE assays,
and correlation coefficients were calculated based on all 44 samples
(Table 1). All of the comparisons produced correlation coefficients
>0.9, indicating a high correlation between the immunoRCA microarray
assay and the other commercial methods. The results indicate that the
specificity and sensitivity of the immunoRCA microarray immunoassay for
allergen-specific IgEs are comparable to those for commercial assays.[FIGURE 1 OMITTED]To demonstrate the lower limit of detection and dynamic range of
the immunoRCA assay, the peanut-positive serum with an ASM score of 6
was serially diluted in negative serum and tested on microarrays and in
the three commercial assays (data not shown). The immunoRCA assay was
linear over a 4456-fold dilution range, and the lower limit of detection
was at least 10-fold lower than the detection limits for the commercial
assays.An important advantage of microarray assays is the ability to
multiplex. The immunoRCA microarray assay is capable of simultaneously
screening hundreds of allergens. Microarrays also include internal
control spots and calibrators, allowing more rigorous standardization of
the results than is possible in other formats. Microarray assays require
<1 nL of allergen extract per test, which allows the use of the more
expensive allergen extracts used for SPT in microarray production. Use
of the SPT extracts may be a factor behind the superior clinical
accuracy reported for the microarray allergen-specific IgE assay (8)
compared with the CAP assay, which uses crude allergen extracts. Another
advantage of microarray assays, which may be of particular value in
pediatric patients, is the requirement for only 10 [micro]L of serum in
these assays. The 10-[micro]L serum volume may allow the use of finger
pricks in place of venipuncture for collection of test samples.
ImmunoRCA allergen-specific IgE microarray assays can provide a
powerful screening tool for allergists. Microarray assays for use in an
allergy clinic can be separated into panels of allergens to be tested
concurrently. The panels can cover allergens falling into similar
categories, such as inhaled allergens, food allergens, or drug
allergens. Arrays corresponding to these panels can be customized to
reflect regional differences in environmental allergens. The immunoRCA
microarray assay uses a 16-well format in which the arrays are separated
by a Teflon mask. The wells can hold 100-400 spots, thus allowing
thousands of assays to be completed per slide. Additionally, the
microarray layout allows the assay to be automated using a Beckman
BioMek liquid-handling robot.ImmunoRCA microarray assays are capable of providing quantitative
results (7). Quantitative allergen-specific IgE assays allow allergists
to accurately monitor immunotherapy techniques, screen infants and small
children for atopic allergen sensitivities, limit the need for oral
challenges with food allergens, and monitor the effectiveness of
allergen avoidance strategies (4). Quantitative microarray assays are
calibrated with calibration curves generated from serial dilutions of
target analytes or dilutions of target analytes immobilized directly on
the surface of each microarray. The advantage to the second approach is
that each microarray contains its own internal calibration curve,
eliminating the effects of variability between arrays. We anticipate
that immunoRCA microarray allergen-specific IgE assays will provide
allergists with quantitative results and more information in a rapid
time frame, increasing cost-effectiveness and the quality of patient
care.We would like to acknowledge Dr. R. Murli Krishna and Mehul Patel
for technical assistance. We thank Drs. Stephen Kingsmore and David
Edgar for expert advice and clinical insight.References(1.) Dolen WK. It is not yet time to stop skin testing, but... . J
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ed. Chichester: Ellis Honaood Limited, .Michael C. Mullenix, [1]* Steve Wiltshire, [1] Weiping Shao, [1]
Gary Kitos, [2] and Barry Schweitzer [1][1] Molecular Staging Inc., 300 George St., Suite 701, New Haven,
CT 06511; [2] Esoterix Inc., 201 Summit View Dr., Suite 100, Brentwood,
TN 37027; * author for correspondence: fax 203-772-5276, e-mail
Table 1. Correlation (a) between microarray assay and commercial
clinical allergen-specific IgE assays.
Correlation coefficient
DPC AlaSTAT
Esoterix Modified RAST
Pharmacia CAP
Cat dander
Short ragweed
Mite (D. farinae)
(a) Correlation coefficients were calculated to compare two data
sets with different units of measurement (9).
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