Red light-controlled polymerase chain reaction

Article abstract of DOI:10.1039/c5cc01805f

A 23-mer DNA "caged" at its 3′-terminus with a 9-anthracenyl moiety was prepared. It can be uncaged in the presence of photosensitizer (In(pyropheophorbide-a)chloride)-containing DNAs (9-12 mers) and upon irradiation with red light. This mixture of DNAs was used to design red-light controlled polymerase chain reaction.

Full text of DOI:10.1039/c5cc01805f

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Red light-controlled polymerase chain reaction
Cite this: DOI: 10.1039/x0xx00000x
A. Meyer,a Margot Schikoraa and A. Mokhira
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
www.rsc.org/
A 23-mer DNA “caged” at its 3’-terminus with a 9- Herein we report on the strategy for caging of primers at the 3’-OH
anthracenyl moiety was prepared. It can be uncaged in the group for the PCR controlled by low power red light (650 nm, 0.29 W)
presence of
a)chloride)-containing DNAs (9-12 mers) and upon We tested two approaches (
irradiation with red light. This mixture of DNAs was used to group of one of the primers was alkylated with a 9-anthracenyl
a
photosensitizer (In(pyropheophorbide- lacking the above mentioned disadvantages of UV-light.
A
and , Figure 1). In both cases 3’-OH
B
design a red-light controlled polymerase chain reaction.
fragment (ON1-AN), whereas another one remained unmodified
(ON3). In approach A conjugate PS-ON2a (PS= In(pyropheophorbide-
Polymerase chain reaction (PCR) is highly sequence specific and
sensitive. It is routinely used in biological applications for
amplification and quantification of DNAs and, in combination with
reverse transcription, of RNAs.1 Moreover, PCR is a key process in
nucleic acid sequencing, cDNA cloning, detection of single nucleotide
polymorphism (SNP)2 and in nanotechnology.3
a)chloride) was designed to bind to the DNA target in proximity to the
modified primer ON1-AN as it is shown in Figure 1. Upon irradiation of
Development of light-sensitive PCR can further broaden the scope of
its applications. For example, Dmochowskii and co-workers4 and later
on Deiters,5 Kuzuya and Komiyama with colleagues6,7 have achieved
photochemical control of PCR by using switchable primers containing
caged nucleotide units. Upon irradiation of these compounds with UV
light, they release activated primers, which support PCR.
Furthermore, Turro and Ju with co-workers have used 3’-caged
nucleoside-5-triphosphates as photoresponsive substrates for PCR in
light-mediated pyrosequencing. In this system, the 3’-OH groups
could be deprotected by irradiation with 355 nm-light.8 Finally, Deiters
and co-workers have developed “caged” Taq polymerase by
introducing a 365-nm-light-responsive tyrosine derivative into the
enzyme.9 In all of these approaches 2-nitrobenzyl-based caging
moieties were applied, which are responsive to UV-light. The latter
trigger can potentially induce undesired side reactions during PCR
including e.g. modification of nucleic acids.10 Moreover, UV-light is
harmful for human eyes and UV-light sources are in general more
expensive than visible light sources. Recently, groups of Heckel11 and
Klán and Wirz12 have described a range of protecting groups sensitive
Figure. 1. Two designs (A and B) of red-light activated primers for polymerase
chain reaction (PCR). PS= photosensitizer, primers: ON1 and ON3, “caged”
primer: ON1-AN, amplified nucleic acid: nucleic acid target.
this construct with red light, which is absorbed by the PS, singlet
oxygen (1O2) is generated in proximity to the 9-anthracenyl moiety
(AN). The 1O2 reacts with the AN inducing its cleavage and release of
functional primer ON1. The rate of the reaction shown in inset
A
(Figure 1) is dependent upon the concentration of the nucleic acid
target.13 In cases when the target is present in small quantities, the
uncaging is expected to be incomplete. In contrast, in the second
to visible light at
to control PCR.
λ<528 nm. However, they have not been yet applied
approach
B, PS-ON2b was designed to bind to ON1-AN. Here the
uncaging reaction is expected to be practically independent of the
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DOI: 10.1039/C5CC01805F
target concentration providing the concentration of primers is was followed by its HPLC purification and MALDI-TOF mass
substantially higher than that of the target. spectrometric identification.† PS-modified conjugates PS-ON2a and
We have used earlier an AN for “caging” siRNAs.14 However, in this PS-ON2b were prepared as previously described.14 All conjugates
known case primary alcohol group (5’-OH) was protected. In used in this paper were >90 % pure according to analytical HPLC
a
contrast, in ON1-AN the AN fragment protects a secondary alcohol (Figures S12, S13).†
group (3’-OH). To test if “uncaging” can in this case also be cleanly In tests of photoresponsive primers we used a synthetically prepared
induced by 1O2 we prepared and studied the reactivity of a model 50-mer DNA as a target (Figure 2). Sequence of this DNA matches a
compound
3
(Figure 2).
part of the sequence of
assembly ON1-AN/Target/PS-ON2a was expected in the buffered
aqueous solution (pH 7.4) of ON1-AN, PS-ON2a and target (design
β-actin-mRNA. At 22°C formation of a stable
A
,
Figure 1). This suggestion is based on high UV-melting points of
duplexes ON1/target (Tm= 73.9 + 0.6 °C) and target/PS-ON2a (Tm= 62
+ 1 °C) at these conditions. We observed that irradiation of ON1-
AN/target/PS-ON2a with red light (650 nm, 0.29 W) for 10 min leads
to conversion of 73 % of ON1-AN into the uncaged primer ON1 (Figure
3A). Further irradiation does not improve the reaction yield
ON1-AN
Target
B
A
PS-ON2b
ON1-AN
Irradiation time
Irradiation time
PS-ON2a
0 min
0 min
ON1
ON1
10 min
5 min
0
20
Time (min)
40
0
10
20
30
Time (min)
Figure 3. A: HPLC traces (monitored by detecting absorbance at 260 nm) of a
mixture of ON1-AN (25 µM), PS-ON2a (1 eq) and Target DNA (1 eq) in annealing
solution (pH 7.4, HEPES 6 mM, KOH 5 mM, KCl 20 mM, MgCl2 0.4 mM) obtained
before and after its 10 min-long irradiation with red light (LED array, 650 nm,
0.29 W). B: HPLC traces of a mixture of ON1-AN (20 µM) and PS-ON2b (1 eq) in
annealing solution obtained before and after its 5 min-long irradiation with red
light.
Figure 2
CCl3CO2H, CH3CN; (c) 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, (i-
Pr)2NEt, CH2Cl2. : The mechanism of the reaction of : A
with 1O2 (steps d-e).
list of conjugates described in this paper and an outline of synthesis of ON1-AN:
(f) reverse DNA synthesis; (g) 1) 1H-tetrazole, 2) Ac2O, pyridine, N-
. A: Synthesis of phosphoramidite 4: (a) anthrone, NaH, DMSO; (b)
B
3
C
substantially, since the photocatalyst PS-ON2a is bleached
simultaneously with the uncaging. Similar effect has been observed
earlier during uncaging of red light activated siRNAs.14 In contrast to
4
,
methylimidazole, 3) I2, pyridine, H2O, THF, 4) CCl3CO2H, CH2Cl2.
design
A, in design B the photosensitizer-containing conjugate PS-
In particular, in the first step compound 115 was alkylated in the
ON2b was selected to be complementary to ON1 rather than to the
template. Since ON2b is rather short (9-mer), ON1-AN/PS-ON2b
duplex was expected to be only partially formed at 22 °C (Tm[ON1/PS-
ON2b] = 28 + 1 °C) thereby enabling quick strand exchange between
the duplex and the target (Figure 1B). Despite the low stability of the
duplex, the residing time of the PS in proximity to the AN seemed to
be sufficient to induce efficient uncaging of the latter fragment upon
irradiation with the red light. In particular, already after 5 min of
irradiation of the ON1-AN/PS-ON2b 89 % of ON1-AN was found to be
presence of anthrone and NaH (step
a) that was followed by cleavage
of 4,4’-dimethoxytrityl (DMT) group at acidic conditions (step
b
). We
observed that the absorbance and emission intensity characteristic for
an anthracene fluorophore (Figures S7, S8) are decreased upon
irradiation of
red light (650 nm, 0.29 W, Figures S7, S8). By using 1H NMR
spectroscopy we confirmed that at these conditions is converted to
anthraquinone and thymidine: 65 % conversion upon irradiation of
(5 mM in CDCl3), PS (0.1 eq.), CF3CO2H (1 %) mixture for 30 min
(Figure S9). In the absence of the acid the reactivity of was reduced
by 1.3 fold that may indicate the involvement of H+ as a catalyst in
conversion of intermediate to (Figure 2B). Under the competitive
conditions the initial rate of uncaging of compound is 1.7 times
3
/ In(pyropheophorbide-a)Cl16 (0.1 eq., PS) mixture with
3
6
3
converted to ON1 (Figure 3B). These data indicate that both designs
and are suitable for primer caging.
A
B
3
Finally, we explored the applicability of “caged” primer ON1-AN in
PCR. In the control experiment, 295-nucleotide-long stretch of cDNA,
5
6
which was obtained by the reverse transcription (RT) from
β-actin
3
mRNA present in the total RNA isolated from HeLa cells (15-150 ng),
was amplified by using ON1 and ON3 as primers, both in the absence
faster than that of previously reported 5’-O-(anthracenyl)thymidine
(Figure S10, S11).14
7
and presence of PS-containing conjugates PS-ON2a (design
A) and
Next,
3 was converted to phosphoramidite 4 (Figure 2A). The latter
PS-ON2b (B). We observed that the amplification was practically
compound was coupled onto the controlled-pore-glass (CPG)-bound
ON1 oligonucleotide, which was assembled by using commercially
unaffected by the latter compounds (Figure S16, S17). Moreover, both
in the absence (trace 1, Cp= 29) and presence of the target (present in
15 ng total RNA, trace 2, Cp= 25) amplification of nucleic acids
occurred (Figure 4A). Melting point analysis of the resulting mixtures
available phosphoramidites for reverse (5’
ꢀ3’) DNA synthesis as
outlined in Figure 2. Final cleavage and deprotection of this conjugate
2 | J. Name., 2012, 00, 1-3
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for the red light-dependent PCR than design
A. For practical reasons it
be preferable to prepare a complete mixture containing all
components required for the PCR and then start the reaction by its
irradiation with red light. We observed that in this case the efficiency
of the amplification is reduced from Cp= 23 to 31 (ꢀCp= +8, trace 3,
Figure 4C) with respect to the case when primers are first uncaged and
then the master mix is added (trace 2). These data may indicate that
components of the master mix inhibit to some extent the uncaging.
Further studies are required to better understand and ultimately
alleviate this effect.
Figure 4.
SYBR Green I fluorescence (
A
: Amplification of cDNA derived from
λ
β-actin-mRNA by using (RT)-PCR;
em= 521 nm, ex= 494 nm) was monitored: (1) ON1
In summary, we developed for the first time “caged” primers for PCR,
which can be efficiently activated by red light (650 nm). These
reagents do not generate primer-dimer side products and exhibit
target amplification after uncaging, which depends upon the
concentration of the target present in the mixture.
λ
(1 eq), PS-ON2a (1 eq), ON3 (1 eq, Cp= 29); (2) the same as (1) except total RNA
(15 ng) was added (Cp= 25); (3) ON1-AN (1 eq), PS-ON2a (1 eq), ON3 (1 eq), total
RNA (150 ng, no amplification detected), the same result was obtained with 15
ng total RNA; (4) the same as (3) except total RNA (15 ng) was added and the
mixture was irradiated for 10 min with LED light source (650 nm, 0.29 W) in the
annealing buffer (Cp= 40); (5) the same as (4) except more total RNA (150 ng) was
added (Cp= 31).
obtained in reactions (1) and (2) from
B
: Fluorescence melting profiles (dF/dT versus T) of products
: RT-PCR of -actin-cDNA: (1) ON1 (1
Notes and references
Friedrich-Alexander-University of Erlangen-Nürnberg, Department of
A.
C
β
a
eq), PS-ON2b (2 eq), ON3 (1 eq), total RNA (150 ng, Cp= 17); (2) ON1-AN (1 eq);
PS-ON2b (2 eq), ON3 (1 eq); total RNA (150 ng) and 10 min irradiation in the
annealing buffer before the reverse transcription (RT) (Cp= 23); (3) the same as
(2) except the irradiation was conducted in the master mix (Cp= 31).
Chemistry and Pharmacy, Organic Chemistry Chair II, Henkestr. 42.,
91054 Erlangen.
†
Electronic Supplementary Information (ESI) available: synthesis and
characterization of new compounds, detailed experimental descriptions,
additional NMR spectroscopic, UV-visible and fluorescence as well as
PCR data. See DOI: 10.1039/c000000x/
(Figure 4B) allowed the conclusion that in the absence of RNA the so
called primer-dimer product was formed, which melts at substantially
lower temperature (Tm= 78 + 1 °C) than the desired cDNA duplex (Tm=
87 + 2 °C). Formation of primer-dimers is an often occurring problem
in the analysis of samples containing low concentrations of nucleic
acids. It can be solved by optimization of primer sequences,
conditions of PCR or using hot start PCR. All of these solutions are
time consuming and costly. We were pleased to observe that in the
presence of “caged” primer ON1-AN, catalyst PS-ON2a and uncaged
1
2
A. Kornberg, T. A. Bake, DNA Replication, 2nd ed.; W.H. Freeman
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J. Sambrook, D. W. Russell, In Molecular Cloning: A Laboratory
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4
5
6
7
8
9
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primer ON3 (design A, Figure 1) no amplification product both in the
absence and presence of the target (15 ng and 150 ng total RNA) was
generated in the dark (trace 3, Figure 4A). In contrast, after irradiation
of this mixture with red light for 10 min in the annealing buffer before
RT-PCR the amplified product corresponding to the cDNA duplex (Tm=
87 + 2 °C) was formed in a concentration dependent manner. In
particular, in the presence of 15 ng total RNA the curve with Cp= 40
was observed (trace 4), whereas in the presence of 150 ng total RNA -
that with Cp= 31 (trace 5). It should be mentioned that the Cp-values
obtained for uncaged primers are substantially smaller than those for
the unmodified ones: Cp= 40 versus 25 for 15 ng RNA and Cp= 31 versus
14 for 150 ng RNA. These data indicate that the uncaging process in
this case was not complete. Analogous effects were observed in
previously reported PCR-system controlled by UV-light.5 As discussed
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earlier the photochemical reaction outlined in design
A is
stoichiometric with respect to the template (Figures 1, 3).13 Since the
template (cDNA) concentration is substantially lower than that of the
primers in the mixtures for PCR, only a small portion of the primer can
be uncaged. To solve this problem, we used primers, whose uncaging
11 C. Menge, A. Heckel, Org. Lett., 2011, 13(17), 4620.
12 P. Šebej, J. Wintner, P. Müller, T. Slanina, J. Al Anshori, L. A. P.
Antony, P. Klán, J. Wirz, J. Org. Chem., 2013, 78(5), 1833.
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14 A. Meyer, A. Mokhir, Angew. Chem. Int. Ed., 2014, 53, 12840.
15 S. J. Martin, J. A. Eisenbarth, U. Wagner-Utermann, W. Mier, M.
is not [cDNA]-dependent (design
B, Figure 1). In particular, we
observed that the amplification of cDNA in the mixture of ON1-AN,
PS-ON2b, ON3 and target (Cp= 23) after 10 min of irradiation was
Heinze, H. Pritzkow, U. Haberkorn, M. Eisenhut, Nucl. Med. Biol.
,
substantially (
previous system under the same conditions (trace 2, Figure 4C).
Therefore, we conclude that design is substantially better suitable
ꢀCp= 8) more efficient than that observed for the
2002, 29, 263.
16 D. Arian, E. Cló, K. V. Gothelf, A. Mokhir, Chem. Eur. J., 2010,
16(1), 288.
B
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