Sequence-specific nucleic acid damage induced by peptide nucleic acid conjugates that can be enzyme-activated

Article abstract of DOI:10.1002/anie.200601681

Recruitment drive: A 12 mer peptide nucleic acid(PNA)-flavin conjugate is prepared. When the PNA moiety is hybridized to its complementary sequence in a 25 mer oligo-2′-deoxyribonucleotide, the flavin moiety can recruit an NADPH:flavin oxidoreductase. This complex allows the activation of molecular oxygen in the presence of NADH and the selective oxidation of the oligonucleotide target (see scheme). (Figure Presented)

Full text of DOI:10.1002/anie.200601681

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thus can be either redox cleaved or hydrolyzed. However, the
great majority of the chemical reactants used in this context to
date are non-natural compounds (such as, transition-metal
complexes, polyamines, photosensitizers) and, as a conse-
quence, such systems in general require an activating
mechanism (e.g., light, strong reductants) unavailable within
the cell. An attractive solution to this problem might be
provided by ODN conjugates carrying a chemical moiety
serving to recruit a cellular enzyme and directing the action of
that enzyme towards selective nucleic acid cleavage. Such a
chemical can be a substrate or an inhibitor of the enzyme. To
our knowledge, there is only one example of such a conjugate
that can be “enzyme activated” illustrating this strategy.[2] It is
provided by studies of ODN-camptothecin (or rebeccamycin)
conjugates in which the chemical moieties are selective
inhibitors of Topoisomerase I.[2] In that case the chemical
group has two roles: 1) by recruiting Topoisomerase I, it
directs its action to the selected sequence; 2) by blocking the
religation step, it ensures the persistence of DNA breaks.
There is no reported example of a PNA conjugate used in this
application. Herein we report a PNA–flavin conjugate 1
(Figure 1) in which the flavin moiety can recruit an
NAD(P)H:flavin oxidoreductase (flavin reductase) allowing
the flavin–enzyme combination to activate molecular oxygen
in the presence of NADH and to selectively damage a
complementary ODN in vitro.
DNA Damage
DOI: 10.1002/anie.200601681
PNAs, which are non-ionic ODN analogues with a non-
natural polyamide backbone, have many advantages over
ODN: 1) they are resistant to nucleases and proteases;
2) they display a very high affinity for complementary DNA
and RNA and can bind to single- and double-stranded DNA
targets.[3] Flavin reductases are ubiquitous enzymes, present in
all living organisms, which catalyze the reduction of free
Sequence-Specific Nucleic Acid Damage Induced
by Peptide Nucleic Acid Conjugates That Can Be
Enzyme-Activated**
Philippe Simon, Jean-Luc DØcout,* and
Marc Fontecave*
Sequence-specific targeting and cleavage of nucleic acids (in
DNA or RNA) can be achieved using oligo-2’-deoxyribonu-
cleotides (ODN) or peptide nucleic acids (PNA) covalently
attached to a reactive chemical.[1] These conjugates combine
the ability to recognize (through the ODN or the PNA) and
react (through the chemical) with a selected sequence, which
[*] Dr. P. Simon, Prof. J.-L. DØcout, Prof. M. Fontecave
DØpartement de Pharmacochimie MolØculaire
UMR 5063 CNRS/UniversitØ Joseph Fourier-Grenoble I
ICMG-FR CNRS 2607
5 Avenue de Verdun, 38240 Meylan (France)
Fax: (+33)4-7604-1007
E-mail: jean-luc.decout@ujf-grenoble.fr
mfontecave@cea.fr
Dr. P. Simon
Figure 1. Structures of the compounds reported herein. Flavin 7 was
prepared in 4steps: a) 6-chlorohexanoic acid, TEA, EtOH, reflux
(53%); b) dioxane/H2O (1:1), pH 7.5, reflux (71%); c) NaNO2, acetic
acid (81%); d) DTT, dioxane/EtOH (1:1), reflux (90%). The arrows on
top of 1’ indicate the sites of oxidation. The bridge marks the sequence
in 1’ that is complementary to the PNA sequence in 1. The orientation
of 1 when hybridized to 1’ is also indicated. Fl indicates the isoallox-
azine ring system of the flavin. The carboxylic acid side chain on N10 in
7 is a highly modified version of the normal flavin ribityl chain.
TEA=triethylamine, DDT=4,4’-dichlorodiphenyl-1,1,1-trichloroethane.
Laboratoire de Chimie et Biochimie des Centres RØdox Biologiques
UMR 5047 CEA/CNRS/UniversitØ Joseph Fourier-Grenoble I
CEA-Grenoble
17 Rue des Martyrs, 38054Grenoble Cedex 9 (France)
Fax: (+33)4-3878-9124
[**] The authors thank Christine Saint-Pierre and Didier Gasparutto
(CEA Grenoble) for expert assistance.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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flavins (riboflavin, flavin mononucleotide (FMN), or flavin
(Vm = 70000, Km = 1.5 mm), thus showing that 1 is well
recognized as a substrate by Fre.
adenine dinucleotide (FAD)) by reduced pyridine nucleotides
(NAD(P)H). Reduced flavins react with oxygen efficiently
thus generating radical and oxidizing species (hydroxyl
radicals and H2O2) potentially reactive towards DNA.[4]
They also efficiently transfer electrons to ferric ions.[5] In a
recent study, flavin reductase was shown to promote oxidative
DNA damage in E. coli cells through reduction of intra-
cellular free iron by enzymatically reduced flavins.[6] This
observation is the basis for studying the PNA–flavin/flavin
reductase combination for selectively destroying a DNA
target.
The most remarkable result is that flavin reductase retains
as much as approximately 25% activity when 1 (10 mm) is
hybridized to 1’ (1 equiv) at 258C (below the Tm value). This
activity remained unchanged upon further addition of 1’. The
1:1’ complex is thus a substrate of Fre and this is in marked
contrast with comparable ODN–flavin conjugates which were
shown to be unable to promote NADH oxidation in the
presence of small excesses of a complementary ODN.[11]
Further studies are required to understand the molecular
basis for such a drastic difference.
There is only one reported synthesis of flavin-tethered
PNA.[7,8] In that case the synthetic procedure allows a given
nucleic base to be replaced at various positions along the
PNA chain by the isoalloxazine ring system of the flavin
which attaches through nitrogen atoms, either N3 or N10.[8] The
novel compounds reported herein, with N10 of the isoallox-
azine ring system linked to the N terminal of the PNA
through a highly modified version of the ribityl chain of
riboflavin, are shown in Figure 1.
The PNA–flavin conjugate 1 was synthesized (see Sup-
porting Information) by coupling the carboxylic acid function
of 7 to the N terminal end of the protected 12-mer PNA 2
attached to the solid support of synthesis using O-(7-
azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluor-
ophosphate (HATU) as coupling agent (Figure 1). After
release and deprotection using trifluoroacetic acid (TFA)/m-
cresol, the resulting conjugate 1 was purified by HPLC on C18
reversed phase and characterized by mass spectrometry and
UV/Vis spectroscopy.
The above result thus made PNA–flavin conjugates good
candidates as DNA (or RNA) damaging agents that can be
enzyme activated. This potential was evaluated in an in vitro
system in which 1 was incubated with a 2-fold excess of 1’ (to
ensure full complexation) in the presence of NADH (5 mm),
various concentrations of FeSO4 and Fre (1 mm) in 50 mm Tris
buffer (tris(hydroxmethyl)aminomethane) pH 7.8. The reac-
tion was carried out in the dark to avoid photoreactions of the
flavin moiety and the reaction mixture was analyzed by gel
electrophoresis after 30 min incubation and piperidine treat-
ment (see Supporting Information). The results are shown in
Figure 2. They demonstrate an interesting selectivity of the
Strong binding of compound 1 to the 25-mer ODN 1’
containing a complementary sequence (Figure 1) and 1:1’
duplex formation were confirmed by melting assays (4 mm,
0.1m NaCl, Tm = 618C).
Figure 2. Phosphoimager picture of a 15% polyacrylamide/7m urea
gel showing the cleavage products of the 25 base-pair target 1’ 32P-
labeled at the 5’-end. Lane 1: Sequencing products (G+A) from 1’ in
formic acid; lane 2: 1:1’ duplex (4 mm with 1’ in 2-fold excess)
incubated in the dark at 378C, pH 7.8 (50 mm Tris buffer) for 30 min
in the presence of NADH (5 mm), flavin reductase (Fre; 1 mm), FeSO4
(40 mm); lane 3: as in lane 2 but without NADH and Fre; lane 4: as in
lane 2 but without any iron; lane 5: as in lane 2 with the addition of
SOD (4 mm) and catalase (0.5 mm); lane 6: as in lane 2 but with 1’
(8 mm) and riboflavin (4 mm) instead of the 1:1’ duplex; see text for
details. The orientation of the complementary chain of the PNA–flavin
1 is also shown.
Flavin reductase activity is generally assayed from the
oxidation of NAD(P)H and spectrophotometrically moni-
tored at 340 nm during aerobic incubation with the enzyme
and a flavin substrate. The enzyme used herein as a model is
the flavin reductase from E. coli named Fre. We have
previously shown that Fre has very broad substrate specificity
since it accepts riboflavin, FMN, or FAD as a substrate.[9] This
indicates that the ribityl chain of a flavin plays almost no role
in its recognition by Fre. The crystal structure of the Fre–
riboflavin complex has been determined and indeed shows
that the protein/substrate interaction almost exclusively
involves the isoalloxazine ring system and not the ribityl
chain located at the surface.[10] Riboflavin analogues with
modified lateral chains at N10 are excellent substrates.[9] The
flexibility is such that ODN–flavin conjugates, with the
isoalloxazine ring system attached to the 5’-end of the ODN
are also substrates.[11] Using increasing concentrations of 1 as
the unique flavin component in the assay mixture and 200 mm
NADH at pH 7.8 and 258C, we observed a saturation
behavior for the enzyme activity. The data could be fitted
with a Vm value of 18000 nmol oxidized NADH/minmgÀ1
protein and a Km value for 1 of 3 mm (data not shown), which
are in the same order-of-magnitude range as the values
obtained with FAD (Vm = 26000, Km = 1 mm) and riboflavin
reaction promoted by the Fre/1 combination. Indeed, cleav-
age occurred mainly at the duplex junction where the flavin is
located. The major product resulted from the oxidation of the
closest guanine residue (G12) within the PNA binding site
and a minor one was observed at the closest guanine (G8) of
the GG sequence adjacent to the duplex-simplex junction
(lane 2). This result is consistent with a flavin-dependent
production of reactive species and a short diffusion of the
latter. The cleavage was both enzyme- and iron-dependent
(lanes 3 and 4, respectively) and was inhibited by addition of
superoxide dismutase (SOD) and catalase (lane 5) or by
exclusion of oxygen. This result suggests that the reaction
proceeds by: 1) reduction of the isoalloxazine ring system of
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[3] P. Wittung, S. K. Kim, O. Buchardt, P. Nielsen, B. Nord›n,
the 1:1’ complex by NADH, catalyzed by Fre; 2) reduction of
O2 by the reduced flavin, generating H2O2 in close proximity
of the target, 3) oxidation of adjacent reactive bases by OHC
radicals (Fenton reaction). The cleavage yield was iron-
concentration dependent but cleavage could be observed with
no addition of iron as a result of the presence of iron
impurities in the buffer. Accordingly, no cleavage could be
observed after treating the buffer with chelating resins
(Chelex) to remove these impurities (lane 4). When riboflavin
was used in place of 1, the selectivity was lost and almost all
bases of the target were oxidized, with guanines (in particular
GG doublets) being, as expected, the most reactive sites
(lane 6). Furthermore, the efficiency of the reaction was
significant with a 20% yield based on the target obtained
after only 30 min reaction.
In conclusion, we report herein the first PNA-based
artificial agent that can be enzyme activated for nucleic acid
oxidation. The PNA–flavin 1 displays two interesting proper-
ties: 1) in complex with a complementary strand, it is
recognized as a substrate by a flavin reductase; 2) under
enzyme-activity conditions, specific and efficient oxidation of
the complementary strand at reactive guanines adjacent to
the flavin moiety occurs. Thus 1 combines DNA damaging
activity, arising from its flavin moiety, and selectivity, arising
from its PNA moiety.
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Future experiments will aim at finding whether such
PNA–flavin compounds might function in vivo by recruiting
cellular flavin reductases to direct their action against a target
sequence. Flavin reductases are ubiquitous enzymes found in
all living organisms and have been shown to promote
intracellular Fenton reactions and DNA damaging in vivo.[6]
Thus the flavin- and iron-dependent DNA damage reaction
studied herein occurs in vivo and the question is whether we
can make it sequence-selective with PNA-flavins as shown
herein in vitro. A limitation might be the very poor cellular
uptake of PNAs in general and thus specific transfection
strategies might be required.[12] On the other hand, since
riboflavin conjugation has been shown to facilitate protein
entry into human cells in culture[13] and PNA conjugation to
lipophilic groups results in improved cell uptake,[12] conju-
gates such as 1 could have better cell-penetrating properties
than the corresponding flavin-free PNA. This is under
investigation.
Received: April 28, 2006
Revised: June 30, 2006
Published online: September 26, 2006
Keywords: DNA damage · enzymes · flavins · oligonucleotides ·
.
oxidoreductases
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