Bioconjugate Chem. 2005, 16, 981−985
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DNA-Directed Coupling of Organic Modules by Multiple Parallel Reductive Aminations and Subsequent Cleavage of Selected DNA Sequences Morten Nielsen,§ Vita Dauksaite,† Jørgen Kjems,† and Kurt V. Gothelf*,§ Department of Chemistry, Langelandsgade 140, and Department of Molecular Biology, C. F. Møllers Alle´, Building 130, Aarhus University, 8000 Århus C, Denmark. Received March 15, 2005; Revised Manuscript Received April 29, 2005
A new method for DNA-directed assembly of organic modules by multiple parallel reductive aminations is presented. Linear oligonucleotide-functionalized modules (LOMs) consist of a rigid oligo(phenylene ethynylene) backbone with two salicylaldehyde termini, and each terminus is conjugated with an oligonucleotide sequence. The stability of the tetrahydrosalen-linked modules toward elevated temperature, low pH, nucleophiles, and metal chelators is studied and compared to the analogous metal-salen-linked modules. A linear oligonucleotide-functionalized disulfide-linked module (LOSM) containing cleavable linkers between the organic module and the two DNA sequences is coupled by DNA-directed reductive aminations to nonmodified LOM modules. This enables selective cleavage of the DNA strands of a central module in a structure consisting of three modules, and the reactions are analyzed by electrophoresis and 32P-labeling of one of the DNA sequences of the central LOSM.
INTRODUCTION
DNA-directed synthesis has in just a few years evolved from chemical ligation to being an advanced tool for controlling chemical reactivity. This is obtained by the encoding of organic compounds with DNA sequences to react only with other compounds connected to complementary sequences (1-3). Liu and co-workers have demonstrated this principle to be applicable to a series of organic reactions ranging from acylation reactions to palladium-catalyzed cross-couplings (4, 5). Several fascinating applications of DNA-directed chemistry have already been reported such as new DNA sequence detection methods (6-8), PNA-templated drug release (9-11), combinatorial synthesis and selection of libraries of potential drug candidates (12), and discovery of new organic reactions (13). We have applied DNA-directed reactions for the assembly and coupling of multiple organic modules in parallel to form macromolecular nanostructures with predetermined connectivity (14, 15). The applied building blocks were rigid linear oligonucleotide-functionalized modules (LOM) and tripoidal oligonucleotide-functionalized modules (TOM) (14, 16). The LOM module shown in Figure 1A consists of an oligo(phenylene ethynylene) backbone and contains salicylaldehyde-derived termini to which two 15-nucleotide DNA sequences are attached (16). In former studies by us (2, 14, 15), and by others (17, 18), DNA-directed couplings of salicylaldehydes have been performed by metal salen formation using manganese acetate and ethylenediamine (EDA) (Figure 1B). DNA-directed formation of Ni-salen (17, 18) and Al* To whom correspondence should be addressed. Tel: (+45) 8942 3907; fax (+45) 8619 6199; e-mail:
[email protected]. § Department of Chemistry. † Department of Molecular Biology.
salen (15) have also been reported, and we have found that a variety of metal salts of Co, Fe, Cu, and U form metal-salen products in DNA-directed couplings. EXPERIMENTAL PROCEDURES
Synthesis of LOMs and LOSMs. The linear oligonucleotide-functionalized modules (LOMs) and disulfidemodified modules (LOSMs) were prepared by incorporation of linear module (LM) phosphoramidites into oligonucleotides by standard automated oligonucleotide synthesis (DNA Technology, Aarhus, Denmark) according to previously published procedures (14-16, 19). The DNA sequences are given in Table 1. Dimerization and Trimerization. A solution (10 µL) of the two or three LOM modules (5 µM each) in 100 mM KCl, 50 mM EPPS (pH 8.0 or pH 5.0) were heated to 60 °C for 5 min and cooled slowly to room temperature in a water bath. The coupling reactions were performed by addition of either 0.25 mM ethylenediamine, 1 mM Mn(OAc)2 (for the salen formation at pH 8.0), or 0.25 mM ethylenediamine and 10 mM NaCNBH3 (for the reductive amination at pH 5.0) and incubation for 2 h at 30 °C. Analysis of the reaction products was performed by electrophoresis at 90 V in 7.5% polyacrylamide gels (30: 1.6) in 50 mM Tricine (pH 8.1) and 8 M urea. Samples were loaded in 8 M urea without heating and addition of dyes. Gels were fixed in 50% (v/v) ethanol, stained with ethidium bromide, and photographed in UV light. Stability Testing. Dimers of LOMs coupled via a Mn-salen bond or via a tetrahydrosalen bond was prepared as described above. Stability tests were performed by subjecting the prepared complexes to heat (75 °C for 1 h), acid (acetate buffer, pH 3.8, 200 mM, 5 µL, 75 °C for 1 h)), amine (MeNH2, 40% aq solution, 2 µL, 75 °C for 1 h), and EDTA (EDTA, pH 7.0, 100 mM, 5 µL, 75 °C for 1 h). Analysis of the reaction products by electro-
10.1021/bc0500793 CCC: $30.25 © 2005 American Chemical Society Published on Web 07/01/2005
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Figure 1. Comparison of DNA-directed dimerization and trimerization of LOMs by metal-salen formation and by reductive amination. (A) Structure of the linear oligonucleotide-functionalized module (LOM). (B) DNA-directed metal-salen formation and tetrahydrosalen formation from the same salicylaldehyde precursors. (C) Analysis of the prerequisites for salen formation and reductive amination on a 7.5% denaturing polyacrylamide gel. Top: Reagents and pH values in the different reaction vessels. Gel: Denaturing PAGE in 8 M urea of LOM-1-LOM-2 dimerizations (lanes 1-5) and LOM-1-LOM-2-LOM-3 trimerizations (lanes 6-8). (D) Stability of LOM-1-LOM-2 dimers linked via a metal-salen complex (MS) and by a tetrahydrosalen moiety (HS) toward heat, acidic condition, amines, and metals; lane 1: LOM-1 monomer; lane 2: LOM-MS dimer; lane 3: LOM-HS dimer; lanes 4 and 5: 75 °C for 1 h; lanes 6 and 7: pH 3.8 at 75 °C for 1 h; lanes 8 and 9: 10 mM methylamine at 75 °C for 1 h; lanes 10 and 11: 10 mM EDTA at 75 °C for 1 h. Table 1. DNA Sequences for the LOM and LOSM Structures modulea
DNA sequence
LOM-1 LOM-2 LOM-3 LOM-4 LOSM-5 LOM-6
5′-AGCGCCTTGTTAGAG-LM-TTAGGTCCTAGTTGT-3′ 5′-AGGAGTAAGCGTGGA-LM-CTCTAACAAGGCGCT-3′ 5′-ACTATTTCCGGCAAC-LM-TCCACGCTTACTCCT-3′ 5′-ATTGATCTAGTTGAT-LM-TGTACATCTACACTT-3′ 5′-AAGTGTAGATGTACA-ss-LM-ss-ACTTCAGTTGGTCGT-3′ 5′-ACGACCAACTGAAGT-LM-CTGTAGACATATGTT-3′
a LM: linear module; LOM: linear oligonucleotide-functionalized module; LOSM: linear oligonucleotide-functionalized diulfide-linked module; ss: disulfide spacer.
phoresis at 90 V in 7.5% polyacrylamide gels (30:1.6) in 50 mM Tricine (pH 8.1), 8 M urea. Samples were loaded in 8 M urea without heating and addition of dyes. Gels were fixed in 50% (v/v) ethanol, stained with ethidium bromide, and visualized by fluorography. Radioactive Labeling. LOSM-5 (5 µM) was labeled with 24.2 pmol of gama-32P-ATP (Amersham, 7000 Ci/ mmol) in 50 µL of T4 polynucleotide kinase buffer using 2 µL of T4 PNK (10 U/µL) for 1h at 37 °C. The labeled oligonucleotide was purified on MicroSpin G-50 columns (Amersham Biosciences). Disulfide Cleavage. Couplings of LOM-4, LOSM-5, and LOM-6 to the desired dimers or trimers were performed as described previously. The disulfide cleavage was performed by addition of tricine buffer (pH 8.1, 2 µL, 500 mM) and tris(2-carboxyethyl)phosphine (TCEP) (pH 7.0, 5 µL, 50 mM) and incubation at 35 °C for 2 h. Traditional analysis of the reaction products was performed by electrophoresis at 90 V in 7.5% polyacrylamide gels (30:1.6) in 50 mM Tricine (pH 8.1), 8 M urea. Samples were loaded in 8 M urea without heating and addition of dyes. Gels were fixed in 50% (v/v) ethanol and stained with ethidium bromide. The wet gel was visualized by fluorography or autoradiography for 15 min and analyzed using Imagequant Software (Biorad).
RESULTS AND DISCUSSION
The metal-salen coupled macromolecular structures obtained in this manner are susceptible to hydrolysis at pH < 6 and other conditions. Consequently, we have investigated the DNA-directed double reductive amination of salicylaldehydes in the presence of EDA to form tetrahydrosalen (Figure 1B). This amine-linked structure is expected to be much more stable than the corresponding imine-linked metal-salens. The tetrahydrosalen is also an excellent ligand for various metals, which will ensure the ordered structure of the linkage between the modules (20). Incorporation of metals in the tetrahydrosalen will also maintain the potential conductivity of the macromolecules (15). The results from the coupling experiments between two LOMs or three LOMs under various conditions for Mnsalen formation and reductive amination are shown in the PAGE gel in Figure 1C. Dimerization reactions of LOM-1 and LOM-2 are shown in lanes 1-5. Mn-salen formation proceeds quantitatively at pH 8, whereas no conversion is observed at pH 5. Importantly, reductive amination also proceeds almost quantitatively and in the presence of Mn(OAc)2. In the absence of reductant and metal, but in the presence of EDA, the expected imine intermediate does not have the stability to withstand the
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Figure 2. DNA-directed couplings and subsequent selective cleavage of DNA sequences. (A and B) Coupling of modules by reductive amination at pH 5 and subsequent cleavage of disulfide-linked DNA sequences by TCEP (color code: blue ) organic module, green: ethylenediamine, curved black lines ) 15-nucleotide DNA sequences). (C) Conformation required for the first steps of the DNAdirected reductive amination. (D) Electrophoresis was performed in 8 M urea, followed by ethidium bromide staining and fluorography. (E) Same gel developed by autoradiography.
reaction conditions or the conditions for denaturing electrophoresis since only starting material is observed at pH 5 (lane 5). The same observation has been made at pH 8 (14). Trimerization of LOM-1, LOM-2, and LOM-3 showed a similar trend (lanes 6-8). To probe the expected increase in stability of the tetrahydrosalen (HS)linked LOM-1-LOM-2 dimer compared to the corresponding Mn-salen (MS), we have submitted the products to various harsh conditions. The MS dimer is relatively stable toward heating at 75 °C for 1 h although some hydrolysis is observed (Figure 1D, lane 4). The HS dimer is inert to these conditions (lane 5). Treatment with acid (pH 3.8) for 1 h at 75 °C completely hydrolyzes the MS dimer whereas the HS dimer remain inert (lanes 6 and 7). Methylamine (10 mM, 75 °C for 1 h) also cleaves the MS dimer, but only partly decomposition of the HS dimer is observed (lanes 8-9). We have observed that the presence of EDTA in buffers leads to decomposition of the MS dimer (lane 10). Probably EDTA is capable of capturing the metal from MS-salen, and the resulting salen decomposes in situ or during gel electrophoresis. This is supported by the observed inertness of the HS dimer toward EDTA (lane 11). The increased stability of the linkages between organic modules in the nanostructures described above encouraged us to investigate the selective cleavage of DNA sequences from macromolecular DNA organic hybrid
assemblies. In a previous report we have described the synthesis and cleavage of disulfide-linked oligonucleotides in the LOSM module (15). The LOSM module contains two (CH2)6-S-S-(CH2)6 spacers between the organic module (LM) and the two 15-nucleotide DNA sequences. It was observed that the Mn-salen linkage in LOM dimers was labile to the reagent used for disulfide cleavage: tricarboxyethylphosphine (TCEP). Al-salen-linked dimers were stable toward TCEP, and we demonstrated that the 15-nucleotide DNA sequences were cleaved off a LOSM Al-salen dimer, but we were unable to identify the organic residue (15). In contrast, tetrahydrosalen-linked LOM dimers were completely stable toward TCEP. We have therefore applied the reductive amination for couplings between LOMs and LOSM to induce a partial cleavage of DNA strands from the products (Figure 2A-E). Since the LOSM module contains a chain of 17 additional atoms between the organic module and the oligonucleotides, the reacting salicylaldehydes are not optimally positioned to form the annealed LOM-LOM couple. This is illustrated in Figure 2C. However, the coupling between LOM-4 and LOSM-5 to give the dimer D1 proceeded in high yield, as it appears from denaturing gel electrophoresis (Figure 2A and 2D, lane 5). The release of two of the four DNA sequences in D1 was performed by cleavage of the disulfides with TCEP. Analysis of this reaction by PAGE
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using ethidium bromide staining and fluorography did not give a clear picture of the outcome of the reaction (Figure 2D, lane 6). We observed a strong band at around 30 nucleotides whereas the band at 15 nucleotides is weak, indicating cleavage of the dimer in LOM and LOSM monomers rather than cleavage of disulfides. It is, however, well-known that short ssDNA sequences bind ethidium bromide poorly and give rise to weak bands (21). The conjugated organic backbone in monomeric LOMs and in structures such as D1 is highly fluorescent, which will contribute to the stronger signal at 30 nucleotides. To obtain a clearer picture of this reaction we have radiolabeled the LOSM module at the 5′-end of the oligonucleotide with 32P and analyzed the reaction by autoradiography (2E, lanes 5 and 6) (21). It is clear from this gel that the radiolabeled 15-nucleotide sequence is released in high yield (2E, lane 6). The mobility of the organic residue strongly indicates that the nonlabeled disulfide-linked DNA sequence has also been released (2D, lane 6). In the second reaction sequence (Figure 2B), LOM-4, LOSM-5, and LOM-6 undergo DNA-directed coupling to form trimer T1 in high yield, and the major product has a mobility in PAGE which corresponds well with a trimer (2D, lane 7). The weaker bands result from slight discrepancies in the stoichiometry between the three species involved. Incubation of T1 with TCEP for 2 h in tricine buffer leads to the major product observed in Figure 2D, lane 8. The low mobility of this product initially led us to believe that the disulfide cleavage had failed. From the autoradiography (2E, lane 8) it is clear, however, that the radiolabeled 15-nucleotide DNA sequence had been cleaved and, as shown in the previous experiment, the nonlabeled disulfide-linked DNA sequence was also cleaved by TCEP. The mobility of the 60-nucleotide product T2 in PAGE was unusual low compared to the other 60-nucleotide product D1 (2D, lane 5). This can, however, be explained by the different environment of the DNA. In product D1 the two central DNA sequences are complementary and, due to the covalent bond between the modules, forms in principle a 30-nucleotides hairpin structure (18), which may only be partly denatured in 8 M urea. Product T2 contains four noncomplementary sequences, and this reduces the mobility in PAGE. It is well-known that, for example, a 30-nucleotide hairpin structure has a much higher mobility in denaturing PAGE than a 30-nucleotide ssDNA sequence. We believe that this is also the reason for the low mobility of T2 compared to D1. Cleaving the DNA sequences of the central module in a trimer and conserving the rest of the DNA sequences is ultimately proving that these structures are indeed held together by covalent bonds. The modular approach using encoded LOMs, LOSMs, and TOMs as shown here and in previous publications (2, 14-16) provides a novel method for assembly of molecular nanostructures. By application of the reductive amination demonstrated here for the coupling between modules, we have been able to construct assemblies that have superior stability compared to the metal-salenlinked structures. If metals are incorporated into the tetrahydrosalen linkage, it will give structures with a rigidity comparable to metal-salens will be formed. The incorporation of metals will also give these macromolecular assemblies properties as potential conductors. The selective cleavage of disulfide groups to release the LOSM DNA sequences is an important new feature of these macromolecules, and the generated thiol groups can be used for further functionalization of the structures or for immobilization on gold surfaces. In particular, the free
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thiols in the terminal positions of this type of macromolecular nanostructure would enable the attachment to electrodes. ACKNOWLEDGMENT
This study was funded in part by the Danish Technical Research Council, the Danish National Research Foundation, and the Carlsberg Foundation. LITERATURE CITED (1) Li, X., and Liu, D. R. (2004) DNA-Templated Organic Synthesis: Nature’s Strategy for Controlling Chemical Reactivity Applied to Synthetic Molecules. Angew. Chem., Int. Ed. 43, 4848-4870. (2) Gothelf, K. V., and Brown, R. S. (2005) A Modular Approach to DNA-Programmed Self-assembly of Macromolecular Nanostructures. Chem. Eur. J. 11, 1062-1069. (3) Summerer, D., and Marx A. (2002) DNA-templated synthesis: More versatile than expected. Angew. Chem., Int. Ed. 41, 89-90. (4) Gartner, Z. J., and Liu, D. R. (2001) The Generality of DNATemplated Synthesis as a Basis for Evolving Non-Natural Small Molecules. J. Am. Chem. Soc. 123, 6961-6963. (5) Gartner, Z. J., Kanan, M. W., and Liu, D. R. (2002) Expanding the Reaction Scope of DNA-Templated Synthesis. Angew. Chem., Int Ed. 41, 1796-1800. (6) Ma, Z., and Taylor, J.-S. (2001) Nucleic Acid Triggered Catalytic Drug and Probe Release: A New Concept for the Design of Chemotherapeutic and Diagnostic Agents. Bioorg. Med. Chem. 9, 2501-2510. (7) Cai, J., Li, X., Yue, X., and Taylor, J.-S. (2004) Nucleic AcidTriggered Fluorescent Probe Activation by the Staudinger Reaction. J. Am. Chem. Soc. 126, 16324-16325. (8) Xiao, Y., Pavlov, V., Naizov, T., Dishon, A. Kotler, M., and Willner, I. (2004) Catalytic beacons for the Detection of DNA and Telomerase Activity. J. Am. Chem. Soc. 126, 7430-7431. (9) Ma, Z., and Taylor, J.-S. (2000) Nucleic Acid Triggered Catalytic Drug Release. Proc. Natl. Acad. Sci. 97, 1115911163. (10) Brunner, J., Mokhir, A., and Kra¨mer, R. (2003) DNAtemplated metal catalysis. J. Am. Chem. Soc. 125, 1241012411. (11) Zelder, F. H., Brunner, J., and Kra¨mer, R. (2004) DNAtemplated catalysis using a metal-cleavable linker. Chem. Commun., 902-903. (12) Gartner, Z. J., Tse, B. N., Grubina, R., Doyon, J. B., Snyder, T. M., and Liu, D. R. (2004) DNA-Templated Organic Synthesis and Selection of a Library of Macrocycles. Science 305, 1601-1605. (13) Kanan, M. W., Rozenman, M. M., Sakurai, K., Snyder, T. M., and Liu, D. R. (2004) Reaction Discovery Enabled by DNA-Templated Synthesis and In Vitro Selection. Nature 431, 545-549. (14) Gothelf, K. V., Thomsen, A. H., Nielsen, M., Clo´, E., and Brown, R. S. (2004) Modular DNA-Programmed Assembly of Linear and Branched Conjugated Nanostructures. J. Am. Chem. Soc. 126, 1044-1046. (15) Brown, R. S., Nielsen, M., and Gothelf, K. V. (2004) Selfassembly of aluminium-salen coupled nanostructures from encoded modules with cleavable disulfide DNA-linkers. Chem. Commun., 1464-1465. (16) Nielsen, M., Thomsen, A. H., Clo´, E., Kirpekar, F., and Gothelf, K. V. (2004) Synthesis of Linear and Tripoidal Phenylacetylene-Based Organic Modules for Application in DNA-programmed Assembly. J. Org. Chem. 69, 2240-2250. (17) Czlapinski, J. L., and Sheppard, T. L. (2001) Nucleic Acid Template-Directed Assembly of Metallosalen-DNA Conjugates. J. Am. Chem. Soc. 123, 8618-8619. (18) Czlapinski, J. L., and Sheppard, T. L. (2004) Templatedirected assembly of metallosalen-DNA hairpin conjugates. ChemBioChem. 5, 127-129.
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