Polymerase synthesis of oligonucleotides containing a single chemically modified nucleobase for site-specific redox labelling

Article abstract of DOI:10.1039/c3cc41438h

Enzymatic construction of single-nucleobase redox-labelled oligonucleotides was developed either based on polymerase incorporation of a single modified nucleoside triphosphate (dNTP) followed by primer extension (PEX) with natural dNTPs or based on PEX with a biotinylated one-nucleotide overhang template, magnetoseparation and the second PEX with a full-length template. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3cc41438h

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Polymerase synthesis of oligonucleotides containing a
single chemically modified nucleobase for site-specific
redox labelling†
Cite this: Chem. Commun., 2013,
49, 4652
Received 25th February 2013,
Accepted 2nd April 2013
a
a
b
b
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Petra Menova, Hana Cahova, Medard Plucnara, Ludek Havran,
Miroslav Fojta*bc and Michal Hocek*ad
DOI: 10.1039/c3cc41438h
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Enzymatic construction of single-nucleobase redox-labelled oligo- bioanalytical applications and that is why we report here a
nucleotides was developed either based on polymerase incorpora- new general enzymatic methodology to prepare them.
tion of a single modified nucleoside triphosphate (dNTP) followed
The methodology was developed and tested for the synthesis
by primer extension (PEX) with natural dNTPs or based on PEX of ssON probes bearing a single redox label3 – either an
with a biotinylated one-nucleotide overhang template, magneto- electroreducible 3-nitrophenyl group or electrooxidizable
separation and the second PEX with a full-length template.
3-aminophenyl3b and ferrocenyl groups.3a Therefore, nitro-
phenyl- (dNNO2TPs), aminophenyl- (dNNH2TPs) and ferrocenyl-
Enzymatic synthesis of base-modified DNA and oligonucleo- ethynyl-modified dNTPs (dNFcTPs, Chart 1)3a,b,9 derived from
tides (ONs) by polymerase incorporation of modified nucleo- all four nucleobases were tested and used as substrates for
side triphosphates (dNTPs) is now an established method1 often polymerase incorporation. The modified dGXTPs were prepared
used for fluorescence,2 redox,3 spin,4 barcode5 and reactive6 according to improved methods (see ESI†). Vent(exo-), Pwo and
labelling, as well as for the protection of DNA.7 Primer extension KOD XL were tested as enzymes in this methodology. For the
(PEX) is the best approach for the synthesis of medium-sized list of sequences used in this study, see Table S1 in ESI.†
ONs bearing several modifications, Nicking Enzyme Amplifica-
The plan was to perform single-nucleotide incorporation
tion Reaction is used for short single stranded ONs (ssONs),8 (SNI) of a modified dNXTP followed by PEX using natural dNTPs
terminal transferase incorporation for 30-tail labelling,9 whereas (Scheme 1a). Therefore, the first task was to optimize the SNI
PCR is used for the synthesis of long DNA duplexes containing with each of the 12 modified dNXTPs to get full conversion to a
a large number of modifications. The PEX method can be one-nucleotide longer primer with possibly minimum excess of
combined with magnetoseparation on streptavidin3 beads to the modified dNXTP (to prevent its further incorporation dur-
produce ssONs.
ing the PEX). After initial optimization, Vent(exo-) was selected
However, in all these methods, the modified dNTP is used as the best enzyme (Pwo was found to digest the template in the
instead of one of the natural dNTPs and, therefore, in all the absence of dNTPs due to exonuclease activity and KOD XL did
newly synthesized stretches, all the copies of this particular not give clean SNI products). Then, general conditions for SNI
nucleobase are fully modified. None of these methods is
suitable for the introduction of a single base-modification at
a specific internal position within the sequence. However,
specifically single-labelled ON probes are needed for most
a Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the
Czech Republic, Gilead & IOCB Research Center, Flemingovo nam. 2,
CZ-16610 Prague 6, Czech Republic. E-mail: hocek@uochb.cas.cz
b Institute of Biophysics, v.v.i. Academy of Sciences of the Czech Republic,
Kralovopolska 135, 61265 Brno, Czech Republic. E-mail: fojta@ibp.cz
c Central European Institute of Technology, Masaryk University, Kamenice 753/5,
CZ-625 00 Brno, Czech Republic
d Department of Organic and Nuclear Chemistry, Faculty of Science, Charles
University in Prague, Hlavova 8, CZ-12843 Prague 2, Czech Republic
† Electronic supplementary information (ESI) available: Additional examples of
monoincorporation of other dNTPs into other sequences, additional figures,
experimental procedures, MALDI spectra and melting temperature curves. See
DOI: 10.1039/c3cc41438h
Chart 1 Structures of modified dNXTPs used in this study.
c
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Fig. 1 Denaturing PAGE analysis of products of polymerase synthesis of ONs
bearing a single modification either by SNI-PEX (a) and (b) (ANO2 or ANH2) or
(c) by an indirect SNI–magnetoseparation–PEX approach (CNO2). Experiments are
supplemented with a primer (p). Lane 1: positive control for SNI (either dATP or
dCTP); lane 2: negative control for SNI (no dNTPs present); lane 3: SNI of dNX
(only dNXTP present); lane 4: negative control for PEX (absence of either dATP or
dCTP); lane 5: positive control for PEX (all natural dNTPs present); lane 6: PEX of
SNI-product containing dNX (all natural dNTPs present). In the case of a sequence
when the modification (c) (CNO2) is followed by the same unmodified nucleotide,
the magnetoseparation of the SNI-product (lane 3) was followed by the PEX
using a longer template to give the final modified ON (lane 6).
primer (to prevent additional incorporation of the second
modified dNXTP), magnetoseparation of the extended primer,
annealing with the full-length template and the final PEX
(followed by magnetoseparation in preparative experiments).
Fig. 1c (and Fig. S5 in ESI†) shows that this procedure gives
clean modified ssON products (which were also isolated by
magnetoseparation and characterized using MALDI).
Scheme 1 Outline of the synthesis of ssON containing a single modification
(a) followed by a different nucleobase, (b) followed by the same but unmodified
nucleobase.
Once having all the 4 modified ONs (ON1–ON4 containing
one dNNO2) in hands, we studied the influence of the modifica-
applicable to any of the 12 modified dNXTPs (freshly dissolved tion on the denaturation temperature (Tm). Table S3 (ESI†)
solutions should be used) were found when using 1.3 equiv. of shows that the nitrophenyl modification destabilizes the
dNXTP at 60 1C for 5 min to obtain in all cases clean products duplexes (DTm = ꢀ0.1 to ꢀ2.2 1C depending on the base-pair).
extended by one-nucleotide (Fig. 1a and b and Fig. S1–S4 in
For any applications of redox labelling in DNA diagnostics,
ESI† – lanes 3). For SNI of modified dAXTPs (which are known3 we need to know the sequence dependence of the redox
to be the best substrates for Vent(exo-) polymerase), much potential. If the sequence dependence is very high, the appar-
lower concentrations of the polymerase (0.004 or 0.02 U mLꢀ1 ent redox potential could directly give information about an
for dANH2TP or dANO2TP, respectively) were used than with all unknown sequence. On the other hand, for multi-potential
other dNXTPs (0.1 U mLꢀ1). After the completion of the SNI, the labelling3c,d of each nucleobase with a different label, the
mix of all four natural dNTPs (1000 equiv. each) was added and requirement is to have the possibly narrowest dispersion of
the polymerase performed the PEX for 20 min to give full-length redox potentials so that the response is additive and the
products (lanes 6 in all gels). When the procedure was per- potentials of different labels cannot interfere.
formed on a biotinylated template, the final magnetoseparation
In order to study electrochemical properties of the mono-
(Scheme 1a) furnished the desired pure single-point modified incorporation products including the sequence dependence, we
ssON which was characterized using MALDI-TOF (see Table S2 have prepared a set of ssONs containing either of the four
in ESI†).
dNNO2 labels in sequence contexts (Table 1). This also confirms
This simple SNI-PEX methodology is generally applicable for that the methodology is truly general in terms of the sequence
the incorporation of a single modification into all sequences of the target ON. The nitro-label was chosen with respect to a
when the modified nucleotide is followed by a different nucleo- high electron yield of its electroreduction (4 electrons per label
base. If the same nucleobase should be present next to the under the given conditions),3b giving rise to a well measurable
modification, the polymerase would tend to incorporate the signal even for a single redox tag among several tens of
second modified nucleotide (even at low concentrations of unmodified nucleotides (Fig. S8 in ESI†). To attain good
dNXTP). Therefore a modified procedure had to be developed reproducibility we performed the measurements using a hang-
for such sequences (Scheme 1b). It consists of the SNI using a ing mercury drop electrode. As it is evident from Table 1, the
biotinylated template only one-nucleotide longer than the apparent potentials of the nitro label primary reduction
c
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Table 1 Potentials of NNO2 reduction in different sequence contexts
Ea/V
potential is relatively low (max. 10 mV differences). These data
support our on-going project of multi-potential redox labelling
for electrochemical detection.3c,d
Ea/V
AAA
CAA
CAC
CAT
GAA
GAC
GAG
GAT
TAA
TAT
ꢀ0.505
ꢀ0.505
ꢀ0.505
ꢀ0.505
ꢀ0.500
ꢀ0.490
ꢀ0.485
ꢀ0.485
ꢀ0.500
ꢀ0.490
TUC
GUC
GUA
TCT
GCT
GCA
TGC
GGC
GGA
ꢀ0.510
ꢀ0.515
ꢀ0.505
ꢀ0.495
ꢀ0.510
ꢀ0.515
ꢀ0.510
ꢀ0.515
ꢀ0.505
This work was supported by the Academy of Sciences of the
Czech Republic (RVO: 61388963 and 68081707) and by the
Czech Science Foundation (P206-12-G151).
Notes and references
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For more details see the ESI.
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4654 Chem. Commun., 2013, 49, 4652--4654
This journal is The Royal Society of Chemistry 2013