Photochemical DNA activation

Article abstract of DOI:10.1021/ol070455u

A new photocaged nucleoside was synthesized and incorporated into DNA with the use of standard synthesis conditions. This approach enabled the disruption of specific H-bonds and allowed for the analysis of their contribution to the activity of a DNAzyme. Brief Irradiation with nonphotodamaging UV light led to rapid decaging and almost quantitative restoration of DNAzyme activity. The developed strategy has the potential to find widespread application in the light-induced regulation of oligonucleotide function.

Full text of DOI:10.1021/ol070455u

ORGANIC
LETTERS
2007
Vol. 9, No. 10
1903-1906
Photochemical DNA Activation
Hrvoje Lusic,† Douglas D. Young,† Mark O. Lively,‡ and Alexander Deiters*,†
Department of Chemistry, North Carolina State UniVersity,
Raleigh, North Carolina 27695, and Center for Structural Biology, Wake Forest
UniVersity School of Medicine, Winston-Salem, North Carolina 27157
alex_deiters@ncsu.edu
Received February 21, 2007
ABSTRACT
A new photocaged nucleoside was synthesized and incorporated into DNA with the use of standard synthesis conditions. This approach
enabled the disruption of specific H-bonds and allowed for the analysis of their contribution to the activity of a DNAzyme. Brief irradiation with
nonphotodamaging UV light led to rapid decaging and almost quantitative restoration of DNAzyme activity. The developed strategy has the
potential to find widespread application in the light-induced regulation of oligonucleotide function.
Many recent discoveries have revealed the multifactorial roles
oligonucleotides play in vitro and in vivo. It has been
demonstrated that they can act as catalysts (ribozymes and
DNAzymes),1,2 sensors (aptamers),3 gene expression plat-
forms (riboswitches and antiswitches),4,5 and gene regulatory
elements (antisense DNA, siRNA, and miRNA).4,6
To study the function of oligonucleotides in a detailed
fashion and to employ them as highly specific biological
research tools, precise control over their activity in a spatial
and a temporal manner is required. In this context, light
represents an ideal control element since it can be precisely
controlled in amplitude, location, and timing thus imposing
spatio-temporal control on the system under study.7 The most
common technique of conveying light-regulation to biological
processes involves the installation of a photoprotecting group
on a biologically active molecule that can be completely
removed via light irradiation. This process, termed “caging”,
has been successfully employed to the light-controlled
activation of small molecule inducers of gene expression,
fluorophores, peptides, and proteins.7 DNA and RNA have
been caged as well, mostly through statistical reaction of
the phosphate backbone of the synthesized or transcribed
oligonucleotide with reactive diazo-derivatives of caging
groups.8 The major disadvantage of this approach is that no
control over the position and number of installed caging
groups can be achieved. Moreover, caging groups are only
installed on the phosphate backbone, not on the heterocyclic
base itself, failing to disrupt Watson-Crick base pairing.
Recently, approaches to the site-specific caging of DNA have
been reported. The introduction of an O-4 caged thymidine
has been successfully applied to the photochemical activation
of transcription and aptamer binding.9 However, due to the
lability of the caging group special DNA synthesis conditions
were necessary. An adenosine modified with a sterically
demanding, photoremovable imidazolylethylthio group has
been used to photochemically activate an 8-17E DNAzyme.10
After irradiation for 8-10 min with short-wavelength UV
† North Carolina State University.
(7) (a) Young, D. D.; Deiters, A. Org. Biomol. Chem. 2007, 5, 999-
1005. (b) Tang, X.; Dmochowski, I. J. Mol. BioSyst. 2007, 3, 100-110.
(c) Goeldner, M.; Givens, R. Dynamic Studies in Biology: Phototriggers,
Photoswitches and Caged Biomolecules; Wiley-VCH: Weinheim, Germany,
2005; p xxvii. (d) Mayer, G.; Heckel, A. Angew. Chem., Int. Ed. 2006, 45,
4900. (e) Dorman, G.; Prestwich, G. D. Trends Biotechnol. 2000, 18, 64.
(f) Lawrence, D. S. Curr. Opin. Chem. Biol. 2005, 9, 570.
‡ Wake Forest University School of Medicine.
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10.1021/ol070455u CCC: $37.00
© 2007 American Chemical Society
Published on Web 04/21/2007

light (254-310 nm) only 30% of RNA cleavage was
observed after a 60 min reaction time. Since the caging group
was installed at C-8, no hydrogen bonding of the adenosine
was disrupted. Reversible switching of DNAzyme activity
was previously achieved through incorporation of diazoben-
zene motifs; however, only a 5- to 9-fold rate modulation
upon irradiation was obtained.11
Our goal was to develop a caging approach that fulfills
all of the following requirements: (a) allows for specific
probing of hydrogen bonding of oligonucleotide bases, (b)
enables introduction of the caged monomer under standard
DNA synthesis conditions, (c) provides a caged oligomer
that is stable to a wide range of chemical and physiological
conditions, and (d) allows for excellent restoration of DNA
activity upon brief irradiation with nonphotodamaging UV
light. Recently, we developed a new caging group (NPOM
) 6-nitropiperonyloxymethyl) that proved to be highly
efficient in the caging of nitrogen heterocycles.12 This group
was specifically designed to solve previous problems as-
sociated with chemical stability or slow decaging rates of
photoprotecting groups on nitrogen atoms. Here, we report
the application of this group to the caging of the thymidine
N-3, thus disrupting an essential hydrogen bond. The
phosphoramidite 1 (Scheme 1) was synthesized in 5 steps
6-nitropiperonyloxymethyl chloride (NPOM-Cl)12 was
achieved in 82% yield (Cs2CO3, DMF, rt) furnishing 3.
Removal of the actetate groups (K2CO3, MeOH, 78%) toward
4 followed by selective tritylation of the primary hydroxy
group (DMTCl, DMAP, pyridine) delivered 5 in 91% yield.
Installation of the phosphoramidite (2-(cyanoethyl)diisopro-
pylchlorophosphoramidite, DCM, DIPEA) was achieved in
80% yield under classical conditions completing the synthesis
of 1.
The stability of 1 to DNA synthesis conditions and its rapid
decaging through irradiation with UV light of 356 nm (ꢀ365
) 6887 cm-1 M-1) was demonstrated (see the Supporting
Information). The quantum yield (φ ) 0.094) for the
photochemical removal of the NPOM group was determined
by 3,4-dimethoxynitrobenzene actinometry.14 With the use
of standard DNA synthesis conditions 1 has been incorpo-
rated at all thymidine positions of the 10-23 DNAzyme D1
providing the mutants D2-D7 (Figure 1). The 10-23
Scheme 1. Synthesis of the Caged Phosphoramidite 1
Figure 1. 10-23 DNAzyme bound to its RNA substrate; thy-
midines are highlighted in red. Wild-type DNAzyme D1 and
DNAzymes D2-D7 having 1 incorporated at various thymidine
positions.
DNAzyme is a highly active and sequence-specific RNA
cleaving deoxyoligonucleotide.2,15 It has been successfully
applied to the suppression of genes in vitro and in model
organisms.16
To probe the necessity of free 3-NH groups in these
thymidine residues for the maintenance of DNAzyme activ-
ity, the RNA substrate 5′-GGAGAGAGAUGGGUGCG-3′
was radioactively 5′-labeled with 32P-ATP and exposed to
the seven DNAzymes D1-D7 in a standard reaction buffer
(100 mM MgCl2, pH 8.2, 15 mM Tris buffer) for 30 min at
37 °C (Figure 2). As expected, the original 10-23 DNAzyme
D1 led to almost complete RNA cleavage. DNAzyme D2
exhibited completely inhibited activity due to the installation
from thymidine starting with the preparation of the known
acetylated thymidine 2 (Ac2O, DMAP, 98%).13 Caging with
(10) Ting, R.; Lermer, L.; Perrin, D. M. J. Am. Chem. Soc. 2004, 126,
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(11) (a) Liu, Y.; Sen, D. J. Mol. Biol. 2004, 341, 887. (b) Keiper, S.;
Vyle, J. S. Angew. Chem., Int. Ed. 2006, 45, 3306.
(12) Lusic, H.; Deiters, A. Synthesis 2006, 2147.
(13) Saladino, R.; Crestini, C.; Occhionero, F.; Nicoletti, R. Tetrahedron
1995, 51, 3607.
(14) (a) Zhang, J. Y.; Esrom, H.; Boyd, I. W. Appl. Surf. Sci. 1999, 139,
315. (b) Pavlickova, L.; Kuzmic, P.; Soucek, M. Collect. Czech. Chem.
Commun. 1986, 51, 368.
(15) Santoro, S. W.; Joyce, G. F. Biochemistry 1998, 37, 13330.
(16) Cairns, M. J.; Saravolac, E. G.; Sun, L. Q. Curr. Drug Targets 2002,
3, 269.
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Org. Lett., Vol. 9, No. 10, 2007

To determine the cleavage rates k of the DNAzymes D1,
D2, and D7, the amount of cleaved RNA was quantified at
nine different time points under single-turnover conditions
through integration (using Molecular Dynamics ImageQuant
5.2) of the corresponding radioactive bands in 15% denatur-
ing TBE polyacrylamide gels with a PhosphorImager (Figure
4).
Figure 2. Cleavage of the RNA substrate for 30 min with the 10-
23 DNAzymes D1-D7 without prior UV irradiation. Conditions:
100 mM MgCl2, pH 8.2, 15 mM Tris buffer, 37 °C, 40 nM
substrate, 400 nM enzyme.
of a single caging group on T12. This was expected, since a
previous mutagenesis study of the catalytic core revealed
this to be an essential residue.17 These experiments also
demonstrated that the least essential thymidine residue is
located at position 16. This was confirmed through the
incorporation of 1 at this position leading to still catalytically
active D3, even in presence of the sterically demanding
caging group. We then probed the tolerance of base pair
mismatches in the substrate recognition domains by caging
the thymidine residues T25, T27, and T29. The resulting
DNAzymes D4-D6 displayed lower activity but still induced
substantial RNA cleavage. Previously, single mismatches
between the RNA substrate and the flanking regions have
led to reduced cleavage activity as well.15 However, selective
installation of three caging groups on T25, T27, and T29 leads
to complete inhibition of RNA cleavage activity in D7,
presumably due to the disruption of multiple Watson-Crick
base paring interactions with the substrate.
Figure 4. Cleavage of the RNA substrate with the wild-type
DNAzyme D1 and the caged DNAzymes D2 and D7 (with and
without UV irradiation). Conditions: 10 mM MgCl2, pH 7.4, 15
mM Tris buffer, 37 °C, 40 nM substrate, 400 nM enzyme. The
cleaved RNA has been normalized and the experiments were
conducted in triplicate.
Subsequently, a more detailed time-course investigation
of the light-activation of D2 was conducted (Figure 3). A
The data were fitted (using Microcal Origin 5.0) with an
exponential decay curve of -ekt,18 and as previously ob-
served, the wild-type DNAzyme D1 showed a high cleavage
rate (kD1 ) 0.242 ( 0.013 min-1) under the assay conditions.
As expected from the results shown in Figure 2, the caged
DNAzymes D2 and D7 displayed no cleavage activity (kD2
) ND and kD7 ) ND), demonstrating that caging group
installation on thymidine can completely abrogate both
catalytic activity and substrate binding. Gratifyingly, brief
irradiation for 1 min (365 nm, 25 W) of the caged
DNAzymes led to restoration of catalytic acitivity of D2
(kD2,UV ) 0.131 ( 0.007 min-1) and D7 (kD7,UV ) 0.129 (
0.011 min-1) to 54% and 53% of the original D1 activity,
respectively. After a 30 min incubation time 80% of the RNA
substrate was cleaved by D1, 73% by irradiated D2, and 55%
by irradiated D7. Thus DNAzymes with an excellent light-
triggered switch have been developed.
In summary, we synthesized a new photocaged nucleoside,
which was incorporated into DNA by using standard
synthesis conditions. This caging approach was then used
to probe the necessity of specific hydrogen bonds for activity
of a DNAzyme, and we found that disruption of a single
H-bond can be sufficient to completely inhibit the enzyme.
Surprisingly, installation of the bulky caging group was
Figure 3. Progressing cleavage of the RNA substrate with the 10-
23 DNAzyme D2 after a 1 min UV irradiation (365 nm). Complete
RNA cleavage is observed after 30 min. Conditions: 100 mM
MgCl2, pH 8.2, 15 mM Tris buffer, 37 °C, 40 nM substrate, 400
nM enzyme.
control experiment of just the RNA substrate exposed to UV
light did not result in any cleaved product. Complete cleavage
of the RNA substrate was achieved within 30 min with use
of the unmodified D1, whereas no cleavage was observed
with caged D2 under identical conditions. However, brief
irradiation with nonphotodamaging UV light of 365 nm (25
W) for 1 min initiated decaging and activation of D2. Figure
3 displays the resulting RNA cleavage with complete
consumption of the substrate by 30 min.
(17) Zaborowska, Z.; Furste, J. P.; Erdmann, V. A.; Kurreck, J. J. Biol.
Chem. 2002, 277, 40617.
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2883.
Org. Lett., Vol. 9, No. 10, 2007
1905

tolerated at several positions within the DNAzyme and the
caging of three thymidine residues was necessary to abrogate
binding to the RNA substrate. Restoration of DNAzyme
activity was achieved through decaging with a brief irradia-
tion of 365 nm UV light (UVA light of this wavelength is
far less toxic to cells than UVB light of shorter wavelength
and is typically considered to be nonphotodamaging7c,19),
providing an excellent on/off switch for oligonucleotide
activity.
gene suppression in model organisms, thus providing power-
ful tools for functional genomics studies.
Acknowledgment. We gratefully acknowledge financial
support by the March of Dimes Birth Defects Foundation
(grant 5-FY05-1215) and the Department of Education
(GAANN Fellowship for D.D.Y.). DNA synthesis was
conducted in the Biomolecular Resource Facility of the
Comprehensive Cancer Center of Wake Forest University,
supported in part by NIH grant P30 CA-12197-30. We also
thank Dr. Richard Pon, University of Calgary, for his advice
on the DNA incorporation of 1.
We believe that the photocaged phosphoramidite 1 will
find widespread application in the light-induced regulation
of oligonucleotide function. Most importantly, caged
DNAzymes could allow for the spatio-temporal control of
Supporting Information Available: Synthesis and ana-
lytical data of 1-5 and D1-D7, enzyme assay protocols,
and enzyme decaging study. This material is available free
of charge via the Internet at http://pubs.acs.org.
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Honigsmann, H.; Jori, G.; Calzavara-Pinton, P. C.; Trautinger, F. J.
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