Reversible photocontrol of deoxyribozyme-catalyzed RNA cleavage under multiple-turnover conditions

Article abstract of DOI:10.1002/anie.200600164

(Figure Presented) Lights, camera, action! Photoswitchable nucleoside analogues containing o-, m-, or p-azobenzenes can be inserted in the catalytic core of RNA-cleaving 10-23 deoxyribozymes by replacing a nonconserved residue (see picture). Irradiation o

Full text of DOI:10.1002/anie.200600164

Communications
RNA Cleavage
DOI: 10.1002/anie.200600164
Reversible Photocontrol of Deoxyribozyme-
Catalyzed RNA Cleavage under Multiple-Turn-
over Conditions**
Sonja Keiper and Joseph S. Vyle*
Light is a highly effective and well-established bioorthogonal
trigger by which the chemical or biochemical reactivities of
nucleic acids can be localized in time and space.[1] Typically,
photoactivation results from the irreversible removal of a
masking group from strategic functionalities and has been
utilized for the construction of DNA arrays, the study of RNA
[*] Dr. S. Keiper, Dr. J. S. Vyle
School of Chemistry and Chemical Engineering
Queen’s University Belfast
David Keir Building, Stranmillis Road, Belfast BT95AG (UK)
Fax: (+44)28-9097-6524
E-mail: j.vyle@qub.ac.uk
[**] We thank Prof. R. J. H. Davies, QUB, for help with the illumination
setup and L. A. Cooke, J. N. McClean, and J. Buick for providing the
starting materials. Prof. A. P. de Silva provided valuable discus-
sions. We gratefully acknowledge financial support by the School of
Chemistry and QUESTOR. J.S.V. was supported by a Sir Henry
Wellcome Commemorative Award for Innovative Research
(050837); S.K. was supported by a postdoctoral fellowship by the
Deutscher Akademischer Austauschdienst.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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cleavage and folding, the triggering of deoxyribozyme or
aptamer activities, and also for the in vivo regulation of gene
expression.[2] Alternatively, reversible optical control of DNA
recognition has been reported with backbone-substituted
azobenzene derivatives.[3] E!Z photoisomerization of azo-
benzenes occurs with high quantum yields at 330–370 nm, is
not particularly sensitive to the environment, is fatigue-
resistant, and leads to large conformational and polarity
changes.[4] Modest discrimination in activities between the so-
called irradiated (mainly Z) and more active dark-adapted
(mainly E) states of papain modified by nonspecific attach-
ment of azobenzenes to surface lysines was reported in 1991.[5]
More recent reports have demonstrated that localization of
azobenzenes at strategic residues of transmembrane pro-
teins[6] or DNA-binding oligopeptides,[7] in which the
E isomer is inactive, enables highly effective activity modu-
lation by light.
To date, a single report of photoswitchable 8–17 deoxy-
ribozyme-mediated RNA cleavage has been made.[8] How-
ever, in this report, only single-turnover behavior with a 200-
fold excess of the deoxyribozymes over the substrate is
described, highly attenuated activities are shown, and the
incorporation of two azobenzene units is required for
significant (up to 5.4-fold) light-induced rate modulation.
Both the 8–17 and 10–23 RNA-cleaving deoxyribozymes were
isolated by in vitro selection from the same library,[9] and have
found application in a wide range of roles, for example, as
sensors for bacterial rRNA, metal ions, and effector sequen-
ces, and for the in vivo regulation of specific mRNA levels.[10]
In particular, 10–23 deoxyribozymes can cleave any target
purine–pyrimidine motif and have wide tolerance for modi-
fications in the binding arms or catalytic core, thus enabling
both substrate affinity and serum stability to be significantly
enhanced by chemical modification.[11,12]
Herein, we report the first photoswitchable RNA cleav-
age by nucleic acid catalysts under multiple-turnover con-
ditions, in which only a single nonconserved residue is
substituted by novel, readily accessible nucleotide analogues.
Cleavage rates are enhanced up to ninefold following
irradiation at 366 nm, thus reaching the rates observed for
the unmodified deoxyribozyme.
We prepared 10–23 deoxyribozymes in which a single
nucleotide (T8) within the catalytic core was replaced by a 2’-
deoxyuridylate analogue. Ortho-, meta-, or para-phenylazo-
benzoyl moieties were appended to this residue through a 2’-
amido linkage (Scheme 1a). This site was chosen as deletion
of T8 or substitution by 2’-O-methyluridine has previously
been shown to only minimally perturb deoxyribozyme
activities.[11] The deoxyribozyme constructs were engineered
with short binding arms to promote product release under
multiple-turnover conditions.
Modified nucleoside precursors were prepared by reac-
tion of the protected 2’-aminonucleoside[13] with the N-
hydroxysuccinimidyl ester of the appropriate azobenzene,[14]
and subsequently phosphitylated under standard conditions.
These precursors were incorporated into model octamers
(ACC1GGTA) and also 10–23 deoxyribozyme sequences
(Scheme 1b) by automated solid-phase synthesis with
extended reaction times for introduction of the modified
Scheme 1. a) Azobenzene-modified uridylates used in this study.
b) The 10–23 deoxyribozymes prepared (FAM=6-fluoresceinyl; cleav-
age site indicated by arrow). c) Photoisomerization of the azobenzene
unit (irr-DR and (d-a)-DR refer to irradiated and dark-adapted 10–23
deoxyribozymes, respectively).
residue. Coupling yields as monitored by trityl release were
greater than 98%. After deprotection under anhydrous
conditions, the oligonucleotides were purified by reversed-
phase HPLC and their identities were confirmed by MALDI-
TOF analysis.
The photoisomerization of oligodeoxynucleotide-
appended azobenzenes under irradiation at 366 nm
(Scheme 1c) was investigated by UV/Vis spectroscopy of
the model octamers. When the photoswitches were in the
thermally stable E configuration, the spectra showed local
maxima at 330 (o-1), 321 (m-1), and 329 nm (p-1) that were
typical of azobenzene p–p* transitions. Irradiation at 366 nm
led to loss of these absorption maxima, with photostationary
states being achieved within 8 min. These results were
reproduced with deoxyribozymes (DRs) appended to azo-
benzene, and the E!Z conversion yields were measured
immediately by HPLC. Peak quantification at 260 nm was
used to determine the level of E and Z isomers either directly
for m-DR and p-DR, or by inference using the model octamer
sequence containing o-1. After irradiation, E!Z conversion
yields of 86% for irr-o-DR, 75% for irr-m-DR, and 61% for
irr-p-DR were thus determined. At 268C the ortho and meta
photoswitches within both the model sequences and the
deoxyribozymes were stable toward Z!E thermal back-
isomerization. In contrast, para photoswitches underwent
more rapid thermal reisomerization at this temperature. In all
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cases Z!E photoisomerization was complete following
The effect of photoswitching upon catalysis by the
azobenzene-conjugated deoxyribozymes was also demon-
strated to be reversible under multiple-turnover conditions
with either 5 mol% (o-D R orm-DR) or 10 mol% (p-DR)
deoxyribozyme (Figure 2). After initial irradiation of deoxy-
irradiation at 435 nm for 2 min.
Both azobenzene-modified and unmodified (wild-type)
deoxyribozymes (wt DRs) catalyzed the site-specific cleavage
of a 13-mer oligoribonucleotide substrate labeled at its
3’ terminus with a fluoresceinyl moiety (FAM) to yield a
labeled hexamer and a 2’,3’ cyclic phosphate-terminated
heptamer. The cleavage reactions were thus resolved, visual-
ized, and quantified in a polyacrylamide gel electrophoresis
(PAGE) assay (Figure 1a). Deoxyribozyme solutions were
exposed to light at 366 nm for 10 min or at 435 nm for 2 min,
and reactions were initiated by the addition of substrate RNA
followed by incubation at 268C in the absence of light. To
assess the cleavage activity of the more thermally labile irr-p-
DR, continuous irradiation at 366 nm was performed during
the assay.
Under multiple-turnover conditions, irradiated azoben-
zene–deoxyribozymes maintained essentially wild-type activ-
ities; thus, irr-o-DR, irr-m-DR, and irr-p-DR showed cleavage
rates of 100, 90, and 50%, respectively (Figure 1b). In
contrast, RNA cleavage rates by dark-adapted (d-a) deoxy-
ribozymes were considerably attenuated. The kirr/kd-a ratios in
this assay were determined to be 9:1 for o-DR and p-DR, and
8:1 for m-DR (Figure 1c). Photocontrol of RNA cleavage by
deoxyribozyme–azobenzene conjugates was also demon-
strated by using an unlabeled RNA substrate and reversed-
phase HPLC analysis.[14] The effects observed in these assays
compare well with the results from the PAGE analyses. Thus,
the relative rates of substrate cleavage by irr-o-DR and irr-m-
DR are both the same as for the unmodified deoxyribozyme
wt DR, and irr-p-DR shows 44% of the wild-type activity.
Dark-adapted deoxyribozymes give significantly less conver-
sion than the irradiated constructs; kirr/kd-a discrimination
factors of 6 for o-DR and 5 for m-DR and p-DR constructs
were observed.
Figure 2. Reversibility of RNA (20 mm) cleavage by 10–23 deoxyribo-
zymes o-DR (1 mm), m-DR (1 mm), and p-DR (2 mm) under multiple-
turnover conditions. Key: * (d-a)-o-DR; * irr-o-DR; ~ (d-a)-m-DR;
~ irr-m-DR; & (d-a)-p-DR; & irr-p-DR.
ribozyme solutions at 366 nm the reactions were initiated by
addition of substrate, and RNA cleavage rates comparable to
those previously described were observed. After 120 min the
reactions were irradiated at 435 nm for 2 min to afford the
dark-adapted deoxyribozymes which gave rise to significantly
retarded reaction rates, although these were higher than those
previously observed using purified E-DRs. Thus, kirr/kd-a
discrimination factors of 3.6 for o-DR, 3.8 for m-DR, and
4.1 for p-DR were observed. This difference might be
accounted for by perturbation of the Z!E photoisomeriza-
tion process[4] in the presence of substrate RNA or kinetic
Figure 1. RNA (20 mm) cleavage by 10–23 deoxyribozymes o-DR, m-DR, p-DR, and wt DR under multiple-turnover conditions (10:1 RNA/
deoxyribozyme). a) PAGE analysis of 3’-FAM-labeled RNA substrate strands (wt: wt DR RNA cleavage; c: control without DR). b) Quantitative
analysis of product formation; key: * (d-a)-o-DR; * irr-o-DR; ~ (d-a)-m-DR; ~ irr-m-DR; & (d-a)-p-DR; & irr-p-DR; ” wt DR. c) krel values for
dark-adapted (open bars) and irradiated (filled bars) deoxyribozymes normalized to the unmodified deoxyribozyme reaction.
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folding traps, which do not respond to this isomerization.[15]
However, the initial cleavage rates were recovered following
irradiation at 366 nm (Figure 2).
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Large differences in the steric demands and hydrophobic
characters of the E and Z isomers of para-azobenzene
residues attached to biomolecules are well-described,[16] but
we are unaware of any other report in which the ortho isomers
give similar activity switching. Preliminary NMR spectro-
scopic investigations indicate that both E and Z isomers of p-1
reside in the C2’-endo furanoside pucker typical of 2’-amido
deoxyribonucleoside analogues and their unmodified con-
geners, and so the active conformation of the catalytic core
containing the photoswitch may be modulated in some other
fashion.
Our demonstration of photomodulated deoxyribozyme-
catalyzed RNA cleavage under multiple-turnover conditions
is of particular interest as the irradiated “on” state maintains
wild-type cleavage rates. The novel analogues described
herein enable incorporation of azobenzene moieties with
readily accessible nucleoside derivatives, which have the
potential to maintain essential base contacts and the biolog-
ical activity of nucleic acids. We envisage that the ability to
reversibly modulate the catalytic RNA cleavage rates of the
10–23 deoxyribozyme by light will add a useful tool to the
repertoire of regulatory biocatalysts. We are currently work-
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Received: January 15, 2006
Published online: April 18, 2006
Keywords: azo compounds · deoxyribozymes ·
.
enzyme catalysis · photoisomerization · RNA
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