A caged ligatable DNA strand break [7]

Full text of DOI:10.1021/ja991300n

J. Am. Chem. Soc. 1999, 121, 11579-11580
11579
Scheme 1
A Caged Ligatable DNA Strand Break
Kaijiang Zhang and John-Stephen Taylor*
Department of Chemistry, Washington UniVersity
St. Louis, Missouri 63130
ReceiVed April 22, 1999
The construction of new DNA molecules for biological
purposes relies heavily on the formation and ligation of sticky
ends terminating in 5′-phosphates and 3′-hydroxyls (Scheme 1).1
Self-assembly of complementary sticky ends followed by ligation
is also a basic step in the construction of unnatural nanoscaled
DNA structures2-4 and in certain approaches to DNA-based
computing.5,6 In multistep DNA nanoconstruction, it is often
advantageous to tie together the ends of the strands of synthetic
intermediates in hairpin loops. The absence of ends helps to
maintain the topology of the intermediates, and makes it possible
to remove unreacted starting material and incompletely ligated
molecules by degradation with exonucleases. Sticky ends are then
generated at the appropriate time in a synthetic sequence with
restriction enzymes. There are, however, inherent limitations to
using restriction enzymes to produce sticky ends. First, most
restriction enzymes produce self-complementary sticky ends not
more than four nucleotides in length, and not the long asymmetric
sticky ends that would be optimal for the stable self-assembly of
unique structures in DNA nanoconstruction.7,8 Seeman and co-
workers have reported a method to add short extensions to sticky
ends, but it is laborious and involves three enzymatic steps.9 A
second problem is that, because of their size and structure,
restriction enzymes are sensitive to the local environment and
may not cleave efficiently or even fail to function for certain
structures.10
One general solution to the problem of generating sticky ends
of any length or sequence would be to cage the end by
incorporating it into a hairpin loop with a photocleavable precursor
to a 5′-phosphate and a 3′-hydroxyl (Scheme 1, bottom). This
strategy would also have the advantage of substantially reducing
the length of the intermediate DNA molecules required in a
synthesis. Though there have been several reports of photochemi-
cally triggered DNA strand breaks,11,12 there have been no
examples of methods for directly phototriggering the simultaneous
formation of 3′-hydroxyl and 5′-phosphate termini. Herein, we
report the design and synthesis of the first building block that
can be used to site-specifically introduce such a caged break into
oligonucleotides, and further demonstrate that the break can be
enzymatically ligated. Such a caged ligatable strand break could
be used to produce sticky ends of any desired length or sequence,
in a structure-independent manner.
The design of a building block for caging a ligatable strand
break was based on two criteria: (1) that it would be sequence
independent and (2) that it would be compatible with standard
DNA synthesis chemistry. We had previously developed a
building block based on o-nitrobenzyl photochemistry that
released a 5′-phosphate upon 366 nm irradiation,12 and simply
needed to add functionality to trigger the release of the 3′-OH.
Whereas it is very difficult to displace 3′-OH’s from negatively
charged phosphodiesters, neutral alkyl phosphotriesters containing
a â-hydroxyl group are known to rapidly release an alkoxide
ligand at pH 7 with a half-life of about 1-2 h at room temperature
via intramolecular displacement by the â-hydroxyl group.13-15
Unfortunately, with a phosphotriester, either of two alkoxide
ligands could be displaced. It was expected, however, that the
corresponding methyl phosphonates, which can only release a
single alkoxide ligand, would react even faster based on the
hydrolysis rates of simple phosphonates in comparison to phos-
photriesters.16 Based on these considerations, we designed the
building block 1 (Scheme 2) that was expected to photochemically
release a 3′-OH and a 5′-phosphate when incorporated into an
oligonucleotide with a commercially available phosphonamidite
2.17
The synthesis of building block 1 is outlined in Scheme 3. To
test the efficiency of photoinduced formation of a 3′-hydroxyl
and 5′-phosphate terminated break, the building block was
incorporated into position 13 of the 21-mer by standard automated
DNA synthesis, and the methylphosphonate of T into position
12. The oligonucleotide was deprotected under conditions com-
patible with methylphosphonate linkages18 and was either 5′-end-
labeled with [γ-32P]ATP and T4 polynucleotide kinase to give
9a or 3′-end-labeled by primer-extension opposite d(CTTAG-
GCACGAGTCAATCTTATAC) with [R-32P]dATP and the Kle-
now fragment of E. coli polymerase I to give 22-mer 9b.19 The
gel-purified end-labeled oligonucleotides were then annealed to
the eventual ligation scaffold 13 to form duplexes.
* To whom correspondence should be addressed. Phone: (314) 935-6721.
FAX: (314) 935-6721 or 4481. email: taylor@wuchem.wustl.edu.
(1) Watson, J. D.; Zoller, M.; Witkowski, G. Recombinant DNA; W. H.
Freeman & Company: New York, 1992.
(2) Seeman, N. C. Acc. Chem. Res. 1997, 30, 357-363.
(3) Niemeyer, C. M. Angew. Chem., Int. Ed. Engl. 1997, 36, 585-587.
(4) Seeman, N. C. Angew. Chem., Int. Ed. 1998, 37, 3220-3238.
(5) Adleman, L. M. Science (Washington, D.C.) 1994, 266, 1021-4.
(6) Maley, C. C. EVol. Comput. 1998, 6, 201-29.
(7) Seeman, N. C. J. Theor. Biol. 1982, 99, 237-47.
(8) Zhang, Y.; Seeman, N. C. J. Am. Chem. Soc. 1992, 114, 2656-63.
(9) Liu, F.; Wang, H.; Seeman, N. C. Nanobiology 1999, 4, 257-262.
(10) Zhang, Y.; Seeman, N. C. J. Am. Chem. Soc. 1994, 116, 1661-9.
(11) Giese, B.; Dussy, A.; Elie, C.; Erdmann, P.; Schwitter, U. Angew.
Chem., Int. Ed. Engl. 1994, 33, 1861-1863.
Irradiation of the duplex containing 5′-end labeled 9a with 365
nm light at pH 7.0 afforded a single major band (91%) that
comigrated with the expected 3′-hydroxyl terminated 12-mer 10a
(Figure 1a). Likewise irradiation of the duplex containing 3′-end
labeled 9b resulted in the formation of a single major band (91%)
that comigrated with the expected 5′-phosphorylated 9-mer 11b
(Figure 1b). In another set of experiments cleavage yields of 96%
and 97% were obtained. The lower mobility bands formed during
irradiation presumably correspond to the intermediates in the
sequential photolysis pathway (Scheme 2). As expected for a
strand break terminating in a 3′-hydroxyl and a 5′-phosphate, the
strand break produced from the 5′-end labeled substrate 9a was
converted by T4 DNA ligase and ATP to a band corresponding
to the expected 20-mer ligation product 12a in 64% yield (Lane
(12) Ordoukhanian, P.; Taylor, J.-S. J. Am. Chem. Soc. 1995, 117, 9570-
9571.
(13) Brown, D. M.; Magrath, D. I.; Todd, S. A. R. J. Chem. Soc. 1955,
4396-4401.
(14) Conrad, J.; Mueller, N.; Eisenbrand, G. Chem.-Biol. Interact. 1986,
60, 57-65.
(15) Pettersson, E.; Bergmark, E.; Rannug, U.; Ehrenberg, L.; Stroemberg,
R. Nucleosides Nucleotides 1995, 14, 753-7.
(16) Kluger, R.; Taylor, S. D. J. Am. Chem. Soc. 1990, 112, 6669-6671.
(17) Agrawal, S.; Goodchild, J. Tetrahedron Lett. 1987, 28, 3539-3542.
(18) Hogrefe, R. I.; Vaghefi, M. M.; Reynolds, M. A.; Young, K. M.;
Arnold, L. J., Jr. Nucleic Acids Res. 1993, 21, 2031-2038.
(19) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular cloning:
A
laboratory manual, 2nd ed.; Cold Spring Harbor Laboratory Press: Plainview,
NY, 1989; Vol. 1.
10.1021/ja991300n CCC: $18.00 © 1999 American Chemical Society
Published on Web 11/30/1999

11580 J. Am. Chem. Soc., Vol. 121, No. 49, 1999
Communications to the Editor
Scheme 2
Figure 1. Photoactivated cleavage (pH 7.0) and ligation of 83 nM (a)
5′-end labeled 21-mer 9a or (b) 3′-end labeled 22-mer 9b in the presence
of an equimolar amount of the 17-mer template 13 (shown below the
figure). Irradiation with 365 nm light was conducted at 0 °C with a hand
held UV lamp (720 µW/cm2 at 3 in.) and aliquots were taken at the
indicated times in minutes. Lane L: Ligation of photocleaved products
using T4 DNA ligase and ATP for 16 h at 16 °C after 32 min of irradiation
and 2 h at room temperature. Lane S: Authentic 5′-[32P]-labeled 10a in
panel a, or 11b in panel b. Lane E: Ligation in the presence of 5 equiv
of nonradioactive 11a in panel a or 10b in panel b. Lane C: Ligation of
authentic 10a and 5 equiv of 11a in the presence of the template. Band
I in lane L of panel b is presumably the 5′-AMP derivative of
d(pCGTGCCTAA) that forms as an intermediate in the ligation reaction.
respectively. The second band is presumably the 5′-AMP deriva-
tive of d(pCGTGCCTAA) that forms as an intermediate in ligation
reactions and can sometimes be detected.20,21 When additional
authentic unlabeled 10b was added, the ligation could be driven
almost completely to the fully ligated product (Lane E). These
results verify that the two oligonucleotides produced in the
photolysis reaction are indeed terminated in the expected 3′-OH
or 5′-phosphate and can be fully ligated under driving conditions.
In summary, we have reported the first caged ligatable DNA
strand break, which could be used to cage DNA sticky ends of
arbitrary length, sequence, and symmetry, as well as other
structures, that would be useful for DNA nanoconstruction, and
possibly DNA computation. Because the breaks are phototriggered
they could also be used to spatially address the assembly and
ligation of DNA. The spontaneous release of an alcohol from a
â-hydroxyphosphonate at neutral pH and room temperature may
also be useful for caging other substrates, or for prodrug design.
DNA strand breaks terminating in a 3′-hydroxyl and a 5′-
phosphate are also important intermediates in the replication,
repair, and recombination of nucleic acids.22,23 The ability to cage
ligatable breaks may also be expected to aid in the study or
application of these processes by affording a method for photo-
triggering the formation of such breaks in vitro and in vivo.
Scheme 3
Acknowledgment. This work was funded in part by NIH Grant
CA40463 and CA75556. Mass spectrometry was provided by the
Washington University Mass Spectrometry Resource, an NIH Research
Resource (Grant No. P41RR0954). The assistance of the Washington
University High Resolution NMR Facility, funded in part through NIH
Biomedical Research Support Shared Instrument Grants RR-02004, RR-
05018, and RR-07155, is also gratefully acknowledged.
L, Figure 1a). In another experiment an 80% yield of ligated
product was obtained. In the presence of added unlabeled 11a
the ligation could be driven to 91% of 12a along with 9% of
what appeared to be a -1 deletion product (Lane E). This minor
side product also appeared in the control ligation reaction between
authentic 10a and 11a (Lane C), but in other ligation reactions
this band was less than 3%. Ligation of the photolysis products
of 3′-end labeled 9b resulted in the expected 21-mer 12b in 36%
yield, along with a second band in 64% yield which migrates
such as an 11-mer (Lane L, Figure 1b). In another experiment,
these two products were produced in 63% and 33% yields,
Supporting Information Available: Experimental procedures as well
as chromatographic and spectroscopic data (PDF). This material is
available free of charge via the Internet at http://pubs.acs.org.
JA991300N
(20) Lehman, I. R. Science 1974, 186, 790-7.
(21) Rossi, R.; Montecucco, A.; Ciarrocchi, G.; Biamonti, G. Nucleic Acids
Res. 1997, 25, 2106-13.
(22) Kornberg, A.; Baker, T. A. DNA Replication, 2nd ed.; W. H. Freeman
and Company: New York, 1992.
(23) Friedberg, E. C.; Walker, G. C.; Siede, W. DNA Repair and
Mutagenesis; ASM Press: Washington, DC, 1995.