Homo-DNA templated chemistry and its application to nucleic acid sensing

Article abstract of DOI:10.1039/c1cc11469g

We have investigated the homo-DNA templated Staudinger reduction of the profluorophore rhodamine azide and have applied this reaction to the detection of natural DNA with a hybrid homo-DNA/DNA molecular beacon. In this system the sensing and the reporting

Full text of DOI:10.1039/c1cc11469g

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COMMUNICATION
Homo-DNA templated chemistry and its application to nucleic acid
sensingw
Matthias Stoop and Christian J. Leumann*
Received 14th March 2011, Accepted 19th April 2011
DOI: 10.1039/c1cc11469g
We have investigated the homo-DNA templated Staudinger
reduction of the profluorophore rhodamine azide and have
applied this reaction to the detection of natural DNA with a
hybrid homo-DNA/DNA molecular beacon. In this system the
sensing and the reporting unit are bioorthogonal to each other
which facilitates sequence design and increases fidelity.
and molecular recognition properties were found to deviate
substantially from that of the natural nucleic acids. While
Watson–Crick A-T and G-C base-pairing as well as anti-
parallel strand alignment in duplexes is maintained there is
no cross-pairing between homo-DNA and natural nucleic
acids due to intrinsic structural differences.3 Thus homo-
DNA is a bio-orthogonal, DNA-like pairing system.
Homo-DNA (Fig. 1) is composed of 20,30-dideoxy-b-D-
glucopyranosyl nucleosides linked in a 40-60-fashion via
phosphodiester units. Its properties have been investigated in
detail by Eschenmoser et al. in the early nineties as part of the
quest on why nature chose DNA as the genetic material.1,2
Although deviating from natural DNA by only one additional
methylene group in the sugar ring its structural, biophysical
DNA templated chemistry, originally investigated in the
context of enzyme free replication of DNA,4 has been widely
used in areas such as the synthesis of complex libraries of
organic small molecules of therapeutic interest,5,6 or in DNA
controlled drug release.7 An obvious application is in the field
of nucleic acid sensing and imaging.8 Attractive are templated
chemical reactions that dequench a fluorophore via removal of
a covalently attached quencher,9–12 or chemically convert a
profluorophore into a fluorophore.13–20 An intriguing asset of
such templated reactions is that they can occur in a catalytic
manner provided that product inhibition is largely reduced.21
While non-ligating templated chemistries are promising in
this context it has also been shown that inclusion of
duplex destabilizing elements during ligation may be a viable
strategy.22,23 This dramatically enhances sensitivity as well as
signal to noise ratios which is indispensable for detecting low
abundance nucleic acid sequences.
While in the case of natural DNA or RNA the sequence to
be recognized is at the same time the template, it would be
highly desirable to chemically separate the specific nucleic
acid recognition event from the signal generation event.
Conceptually this should be realizable with chimaeric
homo-DNA/DNA oligonucleotides in which the DNA
domain is responsible for analyte sensing while the homo-
DNA domain is responsible for signal generation. Such a design
would enable us to keep the signal generation domain as short
as possible which would favor catalysis, while the sensing
domain can be as long as necessary to be statistically unique.
We have shown recently that the bio-orthogonal recognition
properties of homo-DNA can be exploited to reduce false
positive signals in nucleic acid sensing when replacing the stem
part of a molecular beacon by homo-DNA.24 Moreover,
careful sequence design of the stem part becomes obsolete
because no cross-pairing with the natural nucleic acid matrix is
to be expected. In this communication we show that homo-
DNA templated catalytic chemical reactions proceed with at
least equal efficiency compared to DNA and that this reaction
Fig. 1 Homo-DNA templated Staudinger reaction of triphenyl-
phosphine attached to the 60-end of the oligonucleotide probe P1with
the profluorophore rhodamine-110-azide attached to the 40-end of the
oligonucleotide probe P2 on a template.
Department of Chemistry and Biochemistry, University of Bern,
Freiestrasse 3, CH-3012 Bern, Switzerland.
E-mail: leumann@ioc.unibe.ch; Fax: +41 31 631 3422;
Tel: +41 31 631 4355
w Electronic supplementary information (ESI) available: Experimental
part and additional Fig. S1–S7. See DOI:10.1039/c1cc11469g
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Table 1 Homo-DNA and natural DNA sequences used in the
templated Staudinger reductions
used before in the context of natural nucleic acids.20 The
corresponding sequences used in these experiments are
summarized in Table 1.
Entrya
Sequence
Comment
As can be seen from Fig. 2 the homo-DNA templated
Staudinger reaction is inefficient in the absence of any template
or with the template containing no t-gap and shows maximum
efficiency if one homo-t nucleotide functions as a spacer in the
template strand. Variation of the length of the probe strands
from 4–7-mers on template HT2 or HT3 with one t-gap
revealed a direct correlation of signal intensity with Tm which
under the conditions of measurement was highest for the
7-mer probes HP1-7 and HP2-7 on template HT3 (Tm of the
ternary complex 33 1C, Fig. S1 (ESIw)). The homo-DNA
templated reaction is mismatch sensitive. Assembly of HP3-6
(t–t mismatch) and HP1-7 on template HT2 shows substan-
tially reduced fluorescence signal generation compared to the
matched pair (Fig. S2, ESIw).
The Staudinger reduction of the homo-DNA probes HP1-7
and HP2-7 only occurs on the homo-DNA template HT3 and
is completely suppressed if the natural DNA template DT1
(one T-gap) is used (Fig. 3). The inverse experiment in which
heptameric natural DNA probes DP1-7 and DP2-7 were
mixed with the homo-DNA or the natural DNA template
HT3 and DT1, respectively, leads to complementary results
(Fig. S3, ESIw). This clearly shows that no cross-pairing
between the two oligonucleotide systems occurs and that they
are orthogonal to each other.
HT1
HT2
HT3
HT4
HP1-6
HP2-6
HP1-7
HP2-7
HP3-6
DT1
DP1-7
DP2-7
MB1
60-cat agg cac gtg cc-40
60-ata ggc tac gtg c-40
60-cat agg cta cgt gcc-40
60-cat agg ctt acg tgc c-40
60-TPP-gcc tat-40
No gap
1 t-gap
1 t-gap
2 t-gaps
6-mer
6-mer
7-mer
7-mer
60-gca cgt-Rhd-N3-40
60-TPP-gcc tat g-40
60-ggc acg t-Rhd-N3-40
60-gcꢁt cgt-Rhd-N3-40
6-mer mismatch
1 T-gap
7-mer
50-CAT AGG CTA CGT GCC-30
50-TPP-GCC TAT G-30
50-GGC ACG T-Rhd-N3-30
7-mer
60-TPP-agg cac g AAG TTA AGA CCT Chimaeric
ATG cgt gcc t-40
beacon
MBP1
MBI1
60-acg tgc ct-Rhd-N3-40
60-NH2-C6-agg cac gt-40
Beacon probe
Complement to
beacon probe
MBT1
MBT2
MBT3
MBT4
50-T5-CAT AGG TCT TAA CTT-T5-30 Matched target
50-T5-CAT AGG TAT TAA CTT-T5-30 G-A mismatch
ꢁ
50-T5-CAT AGG TTT TAA CTT-T5-30 G-T mismatch
ꢁ
50-T5-CAT AGG TGT TAA CTT-T5-30 G-G mismatch
ꢁ
a
Capital letters: natural 20-deoxyribonucleotides; lower case
letters: homo-DNA nucleotides; TPP: triphenylphosphine; Rhd-N3:
rhodamine-110-azide; C6: C6 alkyl linker; underlined characters:
mismatched bases.
is orthogonal to DNA. In addition we provide an example of
specific DNA sensing with a chimaeric homo-DNA/DNA
molecular beacon.
We compared the efficiency of the templated reduction in
both, the homo-DNA and DNA system. Since the thermal
stabilities of homo-DNA duplexes are higher compared to
DNA in an equal sequence context we searched for conditions
in which both systems exhibited the same Tm. This could be
found for the ternary homo-DNA system HT2 and the probes
HP1-7 and HP2-7 in a low salt buffer (10 mM KCl) and for
the ternary DNA system DT1 with probes DP1-7 and DP2-7
in high salt buffer (500 mM KCl, 50 mM MgCl2). Under these
conditions both complexes showed a Tm of 20 1C. The first
order reaction kinetics of the templated reduction (kobs) was
found to be 8.8 ꢀ 10ꢁ4 sꢁ1 for the homo-DNA system and
To test the potential of homo-DNA templated chemical
reactions we focused on a system which consisted of 13–15-mer
template strands and two probe strands of which one was
equipped with a triphenylphosphine (TPP) unit at the 60-end
(P1) and the other with the profluorophore rhodamine-110-azide
at the 40-end (P2) via suitable linkers (Fig. 1). The template
was designed to contain 0–2 additional homo-T nucleosides
opposite to the junction site to allow for proper accommoda-
tion of the reactive groups. The TPP unit reduces the azide
function to an amine in a Staudinger reaction and has been
Fig. 2 Time course of fluorescence signal development of HP1-6 and
HP2-6 (hexamer probes) in the presence of the homo-DNA templates
HT1,2,4 containing no, one or two t-gaps and in the absence of the
template. Experiments were performed at 35 1C in 50 mM KCl,
10 mM Tris, 3.5 mM MgCl2, pH 8.0; probe conc: 400 nM; template
conc.: 200 nM.
Fig. 3 Fluorescence signal development for the templated reaction of
homo-DNA probes HP1-7 and HP2-7 in the presence of no template,
the homo-DNA template HT3 and the DNA template DT1. Experi-
ments were performed at 16 1C in buffer 2 (50 mM KCl, 10 mM Tris,
3.5 mM MgCl2, pH 7.1) with HP1-7 (800 nM), HP2-7 (400 nM) and
the indicated templates (400 nM).
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7.5 ꢀ 10ꢁ4 sꢁ1 for the DNA system and were thus similar to
each other. We also investigated whether the reaction is
catalytic in template by varying the template concentration
at constant probe concentration. The highest turnover
numbers (120 min) were found with 1% template and were
in the range of 6 (Fig. S4 and S5, ESIw). These values are
similar to those found earlier for PNA probes on a DNA
template with the same Staudinger reduction,16 but less than
those reported for PNA probes on DNA templates using acyl
transfer chemistry.9 It is therefore likely that the lifetime of the
aza-ylide intermediate of the Staudinger reduction which
transiently ligates the two probe strands is limiting turnover.
Next we applied this homo-DNA templated chemistry to a
molecular beacon25 format and constructed MB1, in which the
loop part (15 nt) consisted of DNA and the stem part
(7 base-pairs) of homo-DNA (Fig. 4). The 60-end of the
beacon was equipped with TPP and the corresponding probe
MBP1 with the rhodamine azide. In order to avoid unpaired
homo-DNA single strands we added MBP1 as a duplex with
its complement MBI1, reasoning that this reduces stem strand
invasion of MBP1 in the absence of targets and stabilizes the
non-reporting wing by duplex formation.
As can be seen from Fig. 5 target binding efficiently induces
the templated reduction and leads to a fluorescence signal. The
reaction is mismatch sensitive. A transition mutation (C - T)
as well as transversion mutations (C - A and C - G) in the
same position in the center of the loop produces fluorescence
signals that are substantially weaker compared to the matched
target. We find
a match/mismatch signal ratio of 3.5
(t = 20 min) which is slightly better than that of a normal
DNA beacon but slightly inferior to that of a LNA beacon
under similar conditions.26
In conclusion we have demonstrated that homo-DNA
templated chemistry is at least as efficient as natural nucleic
acid templated chemistry. In addition we have developed a
mismatch selective nucleic acid sensor based on a chimaeric
homo-DNA/DNA molecular beacon in which the reporting
domain is decoupled from the sensing domain. With this each
domain can be designed and optimized separately. Moreover,
the bioorthogonality of the reporting domain is expected to
increase fidelity as it does not interact with the nucleic acid
matrix.
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Fig. 5 Fluorescence signal development for the templated reaction of
the molecular beacon MB1 with the probe MBP1 in the presence of
matched and mismatched DNA targets. Experiments were performed
at 35 1C in buffer 1 (50 mM KCl, 10 mM Tris, 3.5 mM MgCl2, pH 8.0)
with MBP1 (200 nM), MB1 (200 nM), MBI1 (200 nM) and the
indicated targets (1.3 mM).
c
7496 Chem. Commun., 2011, 47, 7494–7496
This journal is The Royal Society of Chemistry 2011