© 2000 Oxford University PressNucleic Acids Research, 2000, Vol. 28, No. 3
Nucleic acid mutation analysis using catalytic DNA
Murray J. Cairns, Andrew King and Lun-Quan Sun*
Johnson and Johnson Research Laboratories, Australian Technology Park, Level 4, 1 Central Avenue, Eveleigh,
NSW 1430, Australia
Received November 23, 1999; Revised and Accepted November 30, 1999
The sequence specificity of the ‘10–23’ RNA-cleaving
discriminate between subtle differences in nucleic
acid sequence in a relatively conserved segment of
the L1 gene from a number of different human papil-
loma virus (HPV) genotypes. DNA enzymes specific
for the different HPV types were found to cleave their
respective target oligoribonucleotide substrates with
high efficiency compared with their unmatched
counterparts, which were usually not cleaved or
cleaved with very low efficiency. This specificity was
achieved despite the existence of only very small
differences in the sequence of one binding arm. As
an example of how this methodology may be applied
to mutation analysis of tissue samples, type-specific
deoxyribozyme cleavable substrates were generated
by genomic PCR using a chimeric primer containing
three bases of RNA. The RNA component enabled
each amplicon to be cleavable in the presence of its
matching deoxyribozyme. In this format, the specifi-
city of deoxyribozyme cleavage is defined by
Watson–Crick interactions between one substrate-
sequence which is amplified during PCR. Deoxy-
ribozyme-mediated cleavage of amplicons generated
by this method was used to examine the HPV status
of genomic DNA derived from Caski cells, which are
known to be positive for HPV16. This method is appli-
cable to many types of nucleic acid sequence varia-
tion, including single nucleotide polymorphisms.
To effectively capitalise on the rapidly expanding nucleic acid
sequence database, there is a need for convenient methods
which can readily discriminate between closely related
sequences. Conventional methods for analysing sequence vari-
ation such as restriction fragment length polymorphisms
(SSCP) (2) and sequencing are either time consuming or
limited by their dependence on recognition sequences for
restriction endonucleases which are effected by the mutation.
Gene-specific amplification techniques such as polymerase
chain reaction (PCR) and ligase chain reaction (LCR) can
detect very small amounts of nucleic acids but often lack the
sensitivity to detect specific point mutations without ancillary
technology such as SSCP or RFLP analysis (3). Allele-specific
oligonucleotide hybridisation can also serve this purpose (4,5)
with much greater flexibility, particularly in a microarray
format which allows parallel verification or simultaneous
screening of many targets in one test. Microarrays can effec-
tively sequence small sections of polymorphic nucleic acids by
providing a different probe for each alternative sequence (6,7).
One of the challenges of this methodology is achieving the
desired level of hybridisation specificity, particularly when
discriminating between two sequences that differ by only a
single base mutation. As these point mutations or single nucle-
otide polymorphisms (SNP) only generate small changes in the
melting temperature of an oligonucleotide duplex, these
systems require fine tuning in order to function effectively. For
example, in a low stringency hybridisation it is very easy to
record false positives, whereas if the stringency is set too high,
a false negative may be indicated. Microarrays supporting
thousands of different oligonucleotides can usually meet this
challenge by brute force interrogation of a SNP at every
possible sequence permutation and with the mutation aligned
at all positions of the probe. In a further development of this
strategy, the hybridisation stringency of an oligonucleotide
array assembled on a semiconductor microchip is controlled
electronically by altering the voltage at small electrodes
embedded in the silicone wafer (8). Subtle differences in oligo-
nucleotide melting temperature (Tm) can also be used to differ-
entiate between SNPs by analysis of the melting curve of a
probe during PCR which can be monitored in real time through
fluorescence resonance energy transfer (9).
In this study we explore the potential of a catalytic DNA
known as the ‘10–23’RNA-cleaving DNA enzyme, as a means
of discriminating between a range of closely relatedsequences.
The 10–23 DNA enzyme or deoxyribozyme was derived by in
vitro selection from a combinatorial library of oligonucleotides
(10). It consists of a conserved catalytic domain flanked by two
substrate binding domains, and has the potential to cleave
RNA at any purine–pyrimidine junction (Fig. 1). The deoxy-
ribozyme, as in the case of oligonucleotide hybridisation,
achieves its target specificity by Watson–Crick interactions
which occur via two substrate-binding domains formed by
arms I and II. However, while oligonucleotide hybrids can
tolerate a certain amount of base mismatch, efficient deoxy-
ribozyme-mediated cleavage can usually only occur when the
deoxyribozyme–substrate heteroduplex is perfectly matched
(11,12). Inaddition tothis level of specificity achieved through
the substrate-binding domains, the 10–23 DNA enzyme can
*To whom correspondence should be addressed. Tel: +61 2 8396 5834; Fax: +61 2 8396 5811; Email: email@example.com
Nucleic Acids Research, 2000, Vol. 28, No. 3
also discriminate by its requirement for an unpaired purine at
the substrate cleavage site followed by a paired pyrimidine.
This flexibility should enable deoxyribozyme-based sequence
analysis to identify almost any polymorphism. To demonstrate
the potential of this approach to sequence recognition we
examined the selectivity of human papilloma virus (HPV)
type-specific deoxyribozymes at a small polymorphic site
within a relatively conserved region of the L1 gene. The
specificity of each deoxyribozyme was determined by
comparing the extent of cleavage on the matched substrate
with cross-reactivity on mismatched substrates. As expected,
only the matched deoxyribozymes were capable of generating
substantial cleavage in the various substrates tested. In order to
translate this RNA cleavage assay into a more accessible
format, we designed a chimeric primer with a three base RNA
sequence corresponding to the cleavage site core. When this
generic primer was used to amplify the target DNA, it gener-
ated a specific cleavable site which enabled deoxyribozyme-
mediated identification of the DNA sample.
MATERIALS AND METHODS
DNA/RNA chimeric oligonucleotides were synthesised and
purifiedbyOligos etc. Otheroligos were made byOligos etc or
Pacific Oligos. The name, sequence and origin of each oligo-
nucleotide are given in Table 1.
The specificity of deoxyribozyme-mediated cleavage was
examined by comparing the extent of cleavage achieved
(during a 1 h incubation) by the various matched and
unmatched deoxyribozyme–substrate combinations. The reac-
tions were performed under single turnover conditions with an
8-fold excess of deoxyribozyme. The chimeric substrate oligo-
nucleotides (1 µM) were 5′-end-labelled prior to the cleavage
reaction with 1 U of polynucleotide kinase (New England
Biolabs) in 60 mM Tris–HCl (pH 7.5), 9 mM MgCl2, 10 mM
dithiothreitol, and 10 µCi of [γ-32P]ATP (GeneWorks) at 37°C
for 30 min and 75°C for 5 min. For each cleavage reaction the
labelled substrate and deoxyribozyme were pre-equilibrated
separately in the reaction buffer (50 mM Tris–HCl, pH 7.5, 10
mM MgCl2) at 37°C for 5 min before being combined to a final
concentration of 50 and 500 nM, respectively. After 60 min the
reaction was stopped by mixing samples with an equal volume
of ice-cold buffer containing 90% formamide, 20 mM EDTA
and loading dye. After reaction, the uncleaved substrate and
products were resolved by electrophoresis on a 10% dena-
turing polyacrylamide gel and analysed using a phosphor-
imager (Molecular Dynamics).
Cell culture and DNA preparation
The HPV16-positive Caski cell line was cultured in DMEM
supplemented with 10% fetal calf serum at 37°C. Genomic
DNA was extracted from the cells using a DNA Extraction Kit
(Stratagene) according to the manufacturer’s instructions.
A type-specific deoxyribozyme cleavable substrate was gener-
ated directly at high copy number by a generic HPV L1 PCR.
The RNA component of the cleavage site was incorporated
into the amplicon by a chimeric primer which contained three
ribonucleotides (DT184). This was 5′-end-labelled prior to
amplification with polynucleotide kinase (as described for the
substrates above) and combined (2 pmol) with a degenerate L1
primer (DT185, 20 pmol) and 10 ng of Caski cell DNA in a
mixture consisting of 50 mM KCl, 10 mM Tris–HCl (pH 8.3),
2.5 mM MgCl2, 1.5 U of AmpliTaq DNA polymerase (Perkin
Elmer), 200 µM each dGTP, dATP, dTTP and dCTP. After 25
cycles at 95°C for 30 s, 50°C for 60 s and 72°C for 90 s, the
PCR product was either purified by 6% native PAGE or used
directly in cleavage reactions.
Figure 1. The 10–23 RNA-cleaving deoxyribozyme. This illustration shows
the secondary structure of a generic enzyme–substrate complex formed by
Watson–Crick interactions between the target RNA sequence and the deoxy-
ribozyme binding domains (both represented by N). The conserved 10–23
catalytic motif is situated between the binding domains (arms I and II) and
bridges the unpaired purine of the purine–pyrimidine cleavage site.
Figure 2. HPV type-specific deoxyribozyme–substrate complexes. The
secondary structure of six HPV type-specific deoxyribozymes with their
corresponding substrate sequences. Each of the substrate sequences are
derived from various HPV types (indicated above each complex) and differ
from each other by polymorphisms contained within a small region
corresponding to the 5′ binding domain of the deoxyribozyme.
Nucleic Acids Research, 2000, Vol. 28, No. 3
Table 1. Substrate, deoxyribozymes and primer oligonucleotides
aThe RNA component of the chimeric substrates are indicated in bold.
bThe conserved 15 base motif of the 10–23 catalytic domain was denoted by lower case.
cPrimer DT185 contained degenerate pyrimidines at four positions such that Y = T+C. A non-degenerate version of
this primer specific for HPV16 was also tested. All oligonucleotides were typed in the 5′→3′ direction.
DT148 ACAGTAACAAAUAATTGATTAHPV18 L1
DT149 ACAGTAACAAAUAGATGATTAHPV11 L1
DT150 ACAGTAACAAAUAACTGATTGHPV31 L1
DT151 ACAGTAACAAAUAGTTGATTAHPV6 L1
DT153ACAGTAACAAAUAGTTGGTTA HPV16 L1
DT160TAATCATCTAggctagctacaacgaTTGTTACTGT HPV11 L1
DT174 TAATCAATTAggctagctacaacgaTTGTTACTGT HPV18 L1
DT175 TAATCAACTAggctagctacaacgaTTGTTACTGTHPV 6 L1
DT177 CAATCAGGTAggctagctacaacgaTTGTTACTGTHPV33 L1
DT178 TAACCAACTAggctagctacaacgaTTGTTACTGTHPV16 L1
DT184 GTATCTACCACAGTAACAAAUA HPV L1
DT185 AAYAATGGYATYTGYTGGHPV L1
DT280 AATAATGGCATTTGTTGGHPV16 L1
Figure 3. Deoxyribozyme cleavage-based sequence analysis. The image contains a 16% polyacrylamide sequencing gel used to resolve end-labelled cleavage
product from the uncleaved substrate. Each synthetic substrate derived from one of six HPV types was incubated with its matching deoxyribozyme and the
unmatched counterparts in the presence of magnesium. The substrate sequence origin for each divided reaction set is indicated at the top of the gel. The deoxy-
ribozymes used in each set were numbered 1–7 and code for (1) no deoxyribozyme, (2) HPV11, (3) HPV18, (4) HPV6, (5) HPV31, (6) HPV33 and (7) HPV16.
Nucleic Acids Research, 2000, Vol. 28, No. 3
Cycle cleavage reaction
To maximise the cleavage extent in a double-stranded target
we used multiple cycles of cleavage and denaturation. In this
reaction purified or unpurified PCR product containing the
cleavable substrate was split into six different tubes and
supplemented with a type-specific deoxyribozyme (1 µM) and
the MgCl2concentration made up to 10 mM. The reaction was
then carried out over 10 cycles of thermal denaturation at 80°C
for 10 s followed by hybridisation and cleavage at 37°C for 5
min. As a control of this cycle cleavage reaction, a static incu-
bation at 37°C was also carried out for the same duration (~80
min) after an initial pre-denaturation at 95°C for 2 min. After
the cycle cleavage reaction each sample was mixed with an
equal volume of formamide loading dye and electrophoresed
on a 10% denaturing polyacrylamide gel. The respective
amounts of cleaved and uncleaved substrate were revealed and
analysed using a phosphorimager.
RESULTS AND DISCUSSION
Six chimeric substrates with sequences derived from a rela-
tively conserved region of the L1 gene in different HPV types
were each challenged with a perfectly matched deoxyribozyme
and five unmatched molecules designed to cleave the alterna-
tive substrates. The sequence of arm I for each type-specific
deoxyribozyme was slightly different, so as to correspond to
the polymorphism in each substrate (Fig. 2). The remaining
binding domain (arm II) and catalytic component of each
deoxyribozyme were identical between HPV types.
In general agreement with earlier studies with other
substrates (11) only the specifically matched deoxyribozyme
was capable of achieving substantial cleavage (Fig. 3). The
most intense reaction was observed in the HPV16 substrate
(lane 42), with 24% cleavage achieved by the appropriately
matched deoxyribozyme after 1 h. Some cross-reactivity with
Figure 4. Substrate sequence amplification and cycle cleavage. Schematic representation of a method for generating a deoxyribozyme cleavable substrate from a
small amount of genomic DNA using PCR and a cycle cleavage reaction. In the first three stages of this procedure, genomic DNA is thermally denatured and used
as a template for amplification with generic HPV primers. These flank a polymorphic region which enables the production of type-specific amplicons. As the
reverse primer contains a 3 bp stretch of RNA (upper case) these amplicons are cleavable in the presence of a deoxyribozyme with an arm sequence complementary
to the polymorphic region. Polymorphic purine and pyrimidine bases are denoted by r and y, respectively. In the later stages of this scheme, the substrate cleavage
efficiency in this double-stranded format is enhanced by thermal cycling.
Nucleic Acids Research, 2000, Vol. 28, No. 3
this substrate was observed with the deoxyribozyme specific
for the HPV6 sequence, with 2% cleavage after 1 h. While this
was not surprising considering that they only differ by a SNP,
the difference in cleavage intensity was large enough for clear
discrimination between the two reactions. Similarly, in addi-
tion to a strong reaction with its matched counterpart (20%),
the HPV11 substrate also experienced a low level of cross-
reaction (1%) with the HPV6-specific deoxyribozyme, which
also differed by a SNP. Despite the similarity to both the
HPV16 and HPV11 substrates, the HPV6 substrate, with 19%
cleavage in 1 h by its specifically matched deoxyribozyme,
was only cleaved to a very small extent by the unmatched
HPV16-specific deoxyribozyme (1%). The other three
substrates did not display any significant reaction with
unmatched deoxyribozymes, including HPV31 and HPV 33,
which only differ by a SNP. However, this may be due in part
to the lower cleavage intensity observed with these reactions,
with only 14 and 9% cleavage by the matched deoxyribozyme,
respectively, after 1 h (Fig. 3, lanes 34 and 19).
The cleavage extents achieved by these HPV L1-specific
deoxyribozymes, while being sufficient to produce an unam-
biguous signal, were in general lower than expected. This is
perhaps partly due to the majority DNA–DNA homoduplex
composition of the enzyme–substrate complex, which has
already been shown to be the least stable hybrid and to have a
lower cleavage efficiency compared with other duplex struc-
tures (13). Earlier investigations of the 10–23 deoxyribozyme
have also shown that there is significant sequence-specific
variation in cleavage efficiency. This variation could usually
be reconciled in terms of the heteroduplex stability predicted
by nearest neighbour analysis (10,11,14). In accordance with
the general performance of the HPV L1 deoxyribozymes, the
predicted duplex stability of the enzyme–substrate complexes
was below average. However, the variation between different
L1 substrates did not follow the predicted hybridisation
stability pattern closely. These differences in activity must
therefore be due to more subtle influences of the sequence
polymorphism than gross helix stability.
Substrate sequence amplification
To effectively harness the capacity of the 10–23 deoxy-
ribozymes to discriminate between chimeric substrates with
similar sequences, we designed a chimeric primer with the aim
of producing a deoxyribozyme-cleavable amplicon by a
generic PCR. The primer introduces the fixed RNA component
and extension produces the variable component as it traverses
the template polymorphism (Fig. 4). We explored the potential
of this system by generating a deoxyribozyme-cleavable
amplicon from the L1 gene of HPV16-positive human cells,
such that it contained the same chimeric HPV16 cleavage site
examined earlier using oligonucleotides. This amplicon was
challenged with the matching HPV16-specific deoxyribozyme
and other unmatched analogues (as in the oligonucleotide
cleavage experiment) to ensure that the activity and specificity
of the reaction was maintained with the substrate in this format
(Fig. 5). The results demonstrated this, with only the HPV16-
specific deoxyribozyme showing cleavage activity. As the
substrate was embedded in a double-stranded PCR product we
found that cleavage extent could be maximised by cycling
between 37 and 80°C. Under these reaction conditions, the
thermal denaturation phases gave the deoxyribozyme multiple
opportunities to compete with the template strand during
hybridisation with the substrate-containing strand. This
dynamic reaction scheme was found to produce more cleavage
product than a static incubation at 37°C (Fig. 5).
The combination of amplification by PCR and deoxy-
ribozyme cleavage analysis of polymorphisms provides a
convenient means of discriminating between subtle differences
in genomic DNA. As the 10–23 deoxyribozyme can cleave
almost any purine–pyrimidine junction, this configuration will
be able to accommodate most mutations, particularly SNPs,
many of which would not have been amenable to conventional
RFLP analysis due to the lack of appropriate restriction endo-
nuclease recognition sequences.
In conclusion, the 10–23 deoxyribozyme was shown to be
capable of discriminating between a series of closely related
substrate sequences derived from the L1 gene of various HPV
types. The utility of this deoxyribozyme, which has the poten-
tial for cleaving a broad spectrum of sequences with very high
specificity under simulated physiological conditions, was then
expanded by coupling it with PCR in a method described as
substrate sequence amplification. This system should enable
the deoxyribozyme cleavage approach to be accessible to
almost any nucleic acid sequence, even those in low abun-
dance, provided they are receptive to amplification by PCR.
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Figure 5. Substrate amplification and cleavage. This image was derived from
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left side of the gel. Each cleavage reaction was carried out both at a constant
37°C (static) and by thermal cycling between 80 and 37°C (cycle cleavage) as
indicated at the top of the gel.
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