PNA-based reagents for the direct and site-specific synthesis of thymine dimer lesions in genomic DNA

Article abstract of DOI:10.1021/ja056358i

An acetophenone containing PNA-based reagent was designed for the direct and site-specific synthesis of a cis-syn thymidine dimer lesion in genomic DNA. Copyright

Full text of DOI:10.1021/ja056358i

Published on Web 01/17/2006
PNA-Based Reagents for the Direct and Site-Specific Synthesis of Thymine
Dimer Lesions in Genomic DNA
J. Carsten Pieck, David Kuch, Friederike Grolle, Uwe Linne, Clemens Haas, and Thomas Carell*
Department of Chemistry and Biochemistry, Ludwig-Maximilians UniVersity, Butenandtstr. 5-13,
83177 Munich, Germany
Received September 15, 2005; E-mail: thomas.carell@cup.uni-muenchen.de
Scheme 1a
UV irradiation of cells furnishes large numbers of mutagenic
cyclobutane pyrimidine dimer lesions (CPD, dTddT) as a conse-
quence of [2π + 2π] cycloaddition reactions between adjacent
thymidines.1-4 In recent years, the need for DNA containing such
lesions at defined sites has steadily grown5 in order to allow
biochemical,6 crystallographic,7 and cellular studies.8 These are
needed to learn how DNA lesions induce mutations and how cells
remove lesions from the genome.9 Particularly, DNA with dTddT
lesions is needed because they are repaired by various pathways,
shifting them into the center of DNA repair.10 The chemical
synthesis of DNA containing dTddT dimers is a time-consuming
process. It requires synthesis of a dimer lesion phosphoramidite
building block,11 chemical synthesis of DNA using this building
block, purification of the prepared DNA, and, if required, ligation
of the DNA into a larger DNA fragment. Alternatively, small DNA
fragments with one dTpdT dinucleotide site can be irradiated in
the presence of acetophenone to give dTddT containing DNA and
with side products that need to be separated by HPLC.12,13 Both
methods are labor intensive, and it is difficult to create DNA strands
with more than one thymine dimer.14
We thought that it would be a tremendous advantage if reagents
could be created for the synthesis of a defined lesion in a defined
position directly in large DNA strands or even in genomic DNA.
For dTddT, the incorporation of an acetophenone into peptide
nucleic acids (PNA), which are able to bind tightly and sequence-
specifically to complementary DNA sequences, might allow us to
achieve this goal.
Two potential problems had to be solved. First, energy transfer
from the light-excited acetophenone in the triplet state to the dTpdT
dinucleotide must be faster than H-atom abstraction from DNA,
which would give reactive DNA radicals.15 Second, energy transfer
is a rather long-range process, raising the question of how specific
such reagents could potentially act.16
Syntheses of the acetophenone PNA building block and of the
PNA strands needed for this study are depicted in Scheme 1. First,
4-methylacetophenone (1) was transformed via Wohl-Ziegler
bromination (2), bromine to cyanide exchange (3), and hydrolysis
of the cyanide group into compound 4. Reaction of 4 with the Fmoc-
and t-butyl-protected PNA backbone molecule 5 furnished, after
acidic cleavage of the t-butylester, the acetophenone PNA monomer
(7), denoted as A in Scheme 1. Synthesis of the peptide nucleic
acid strands PNA8-12 needed for this study was performed using
a reported Fmoc synthesis protocol for peptide nucleic acids.17,18
Two lysine residues at the N- and C-termini ensured excellent
solubility (K).
We first analyzed the best position of the acetophenone relative
to the dTpdT site to optimize the energy transfer step. To this end,
the four PNA strands 8-11 were hybridized to the corresponding
DNA strands (DNA1 for PNA8, -10, -11 and DNA2 for PNA9) to
give double strands, in which the A building block is (i) positioned
a Conditions: (a) i NBS, AIBN, MeCN, 1.5 h, 90 °C, 95%; ii NaCN,
dioxane, H2O, 2 h, reflux, 67%; iii H2O, AcOH, H2SO4, 1.5 h, reflux; iV 5,
TBTU, HOBt, DIEA, DMF, 80 min, rt, 97%; V TFA/H2O (95:1), 1 h, rt,
94%; (b) PNA (with A ) acetophenone building block) and DNA (with
TT ) potential thymine dimers) strands.
opposite the 3′dT of the dTpdT dinucleotide (PNA9:DNA2); (ii)
positioned opposite the 5′dT (PNA8:DNA1); (iii) positioned so that
two A units face the dTpdT dinucleotide (PNA10:DNA1); and (iv)
positioned so the A unit is shifted one nucleobase toward the 5′-
end in order to allow the dTpdT to form two intact base pairs
(PNA11:DNA1). Irradiation of these four PNA:DNA double strands
(20 µM PNA:DNA duplex, 10 mM NaCl, 10 mM, phosphate buffer
pH ) 7.4) with λ ) 340 nm was performed to induce dimer
formation. The reaction mixture was analyzed before and after 3 h
of irradiation by reversed-phase HPLC. Whereas irradiation of DNA
with just acetophenone gave rise to a variety of products, irradiation
of all PNA:DNA duplexes gave a clean conversion of the DNA
strands to new DNA products of unchanged molecular weight as
determined by MALDI-TOF. Addition of DNA photolyase enzyme,
which specifically cleaves the naturally occurring cis-syn dTddT
dimers back into the dTpdT dinucleotides, converted the product
strands back into DNA1 and DNA2, respectively, proving that only
cis-syn dTddT dimers and no other products are formed during
the experiment. Most interesting, however, is the chemical yield
of the reaction. The best results were obtained with the duplex
PNA8:DNA1, in which the A building block faces the 5′dT of the
dTpdT site. Irradiation for only 3 h converted DNA1 into the
corresponding dTddT dimer containing DNA strand in yields
between 60 and 80%.
To investigate the selectivity of the method, we prepared the
PNA reagent PNA12, hybridized the reagent to a 51mer (DNA3)
with two dTpdT sites (Scheme 2), irradiated the complex for 3 h,
added the fully complementary DNA counter strand DNA4 (5′-
GCG-CAT-ACG-TGT-ATA-CTT-ACA-TGT-ATA-TAA-TAT-
ACG-CGC-AAC-GTA-CTC-GCC-3′) to form the DNA3:DNA4
duplex, and cut the duplex with the thermophilic restriction enzyme,
BstU1, into a 15mer and a 36mer DNA strand. The 15mer and the
36mer were separated by rp-HPLC and analyzed by agarose gel
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10.1021/ja056358i CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
Scheme 2a
Figure 1. Schematic illustration of generating and analyzing a defined CPD
lesion (red stretch in the plasmid strand) in genomic M13 mp 18 ss DNA.
specifically the targeted sequence in a large genomic DNA fragment
in order to generate selectively upon irradiation the desired cis-
syn dTddT lesion. In summary, we have created novel reagents
that allow synthesis of cis-syn dTddT lesions directly in single-
stranded DNA and even in large genomic DNA.
a Procedure to create a CPD lesion (red) in a 51mer DNA. It is possible
to generate the CPD lesion in only one of the two TT sites, which is analyzed
by subsequent enzymatic digestion and HPLC-MS/MS.
Acknowledgment. We thank the Deutsche Forschungsgemein-
schaft (DFG), the Volkswagen Foundation, the EU Marie Curie
Training and Mobility Program (CLUSTOXDNA), and the Fonds
der Chemischen Industrie for generous financial support of this
research. We thank D. Hammond for proofreading the manuscript.
electrophoresis. The strands were then fully digested with an
enzyme mixture containing nuclease P1, calf spleen phosphodi-
esterase II, alkaline phosphatase (CIP), and snake Venom phos-
phodiesterase I. The two digests were analyzed by HPLC-MS/
MS (Scheme 2). The mass spectra show the peaks for the canonical
nucleobases dA, dC, dG, and dT. Only for the small fragment was
a large additional signal for a dTddT dimer at m/z ) 545 recorded.
The fragmentation pattern of this signal was analyzed by HPLC-
MS/MS and proven to be identical with reported data for a cis-
syn dTddT, proving the correct stereochemistry.13 The results show
that the PNA reagents bind specifically to the wanted sequence in
the DNA strand in order to specifically direct the formation of cis-
syn dTddT. Formation of other lesions, such as (6-4) photoad-
ducts, or trans-syn photo products can be ruled out based on the
mass fragmentation data.
We finally investigated the ability to form site-specifically a dTd
dT lesion directly in genomic DNA. This was tested with a M13
mp 18 phage genome (7249 nucleobases), which was prepared in
the form of a cyclic single strand (Figure 1). The PNA reagent
PNA13 was prepared and hybridized to the genomic DNA, and
the PNA:DNA ds was irradiated for 3 h. The PNA was then
removed using reversed-phase HPLC at 55 °C. A biotin-labeled
oligonucleotide (DNAB, 5′-TCG-CCA-TTC-AGG-CTG-CGC-AAC-
TGT-TBGG-GAA-GGG-CGA-TCG-GT-3′ with TB ) biotin-labeled
thymine) binding to the same sequence containing the potential
thymine dimer was added and hybridized. The construct was bound
to magnetic particles derivatized with streptavidin. Two restriction
enzymes, Bgl1 and PVu1, were used to cut the immobilized
construct into a small 28mer and a large 7221mer DNA piece. The
small and large DNA fragments were spotted onto a positively
charged nylon membrane and treated with a specific anti cis-syn
dTddT dimer antibody and a second antibody, HRP-labeled, to
recognize the first one. As depicted in Figure 1, the chemilumi-
nescence reaction is clearly limited to the spot containing the 28mer
DNA, showing that the PNA reagent was able to recognize
Supporting Information Available: Experimental protocols and
characterization data. This material is available free of charge via the
Internet at http://pubs.acs.org.
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