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Chloroacetamide-Linked Nucleotides and DNA for Cross-Linking with Peptides and Proteins Agata Olszewska,† Radek Pohl,† Marie Brázdová,‡ Miroslav Fojta,‡,§ and Michal Hocek*,†,∥ †
Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Gilead Sciences & IOCB Research Center, Flemingovo namesti 2, 166 10 Prague 6, Czech Republic ‡ Institute of Biophysics, Czech Academy of Sciences, Kralovopolska 135, 612 65 Brno, Czech Republic § Central European Institute of Technology, Masaryk University, Kamenice 753/5, CZ-625 00 Brno, Czech Republic ∥ Department of Organic Chemistry, Faculty of Science, Charles University in Prague, Hlavova 8, 12843 Prague 2, Czech Republic S Supporting Information *
ABSTRACT: Nucleotides, 2′-deoxyribonucleoside triphosphates (dNTPs), and DNA probes bearing reactive chloroacetamido group linked to nucleobase (cytosine or 7deazadaenine) through a propargyl tether were prepared and tested in cross-linking with cysteine- or histidine-containing peptides and proteins. The chloroacetamide-modifed dNTPs proved to be good substrates for DNA polymerases in the enzymatic synthesis of modified DNA probes. Modified nucleotides and DNA reacted efficiently with cysteine and cysteine-containing peptides, whereas the reaction with histidine was sluggish and low yielding. The modified DNA efficiently cross-linked with p53 protein through alkylation of cysteine and showed potential for cross-linking with histidine (in C277H mutant of p53).
B
able for polymerase synthesis of reactive ON or DNA probes which react with cysteine or cysteine-containing peptides or proteins through thiol conjugate addition to the Michael acceptor group.31 The vinylsulfonamide (VS) can also be introduced postsynthetically via CuAAC reaction.32 In both cases, the cross-linking of the VS-modified DNA with protein (p53) required the proximity effect of the specific DNA recognition.31,32 In order to extend the portfolio of reactive groups suitable for ON or DNA modifications and, in principle, be able to also address other amino acid side-chains, we report herein the synthesis and bioconjugation of nucleotides and a DNA bearing chloroacetamide (CA) group linked through a propargyl tether to nucleobases. Chloroacetamide is a mild electrophile which has been reported to react with both cysteine and histidine in cross-linking of diverse (bio)molecules with proteins.33−38
ioconjugation of nucleic acids with peptides or proteins has found many applications in medicinal chemistry, chemical biology, and nanotechnology.1−4 The peptides or proteins can easily be connected to either the 3′- or 5′-terminus of an oligodeoxynucleotide (ON)1−4 but often it is desirable to attach them to nucleobases to place them into the major groove and to facilitate the control of positions of modifications. Due to the inherent difficulties of direct incorporation of peptide- or protein-linked nucleotides, synthesis of ONs containing reactive group capable of bioconjugation or cross-linking to protein has the potential to be a feasible approach. Base-modified nucleotides bearing diverse chemically reactive groups (e.g., aldehyde,5−7 alkyne,8−11 alkene,12−15 diene,16 azide,17−19 or tetrazole20) have been incorporated into DNA chemically or enzymatically and have subsequently been further modified by reductive amination,6,7 CuAAC click reaction,8−11 Diels−Alder reactions,14−16 or Staudinger ligation21 providing diverse labeled ONs or DNA-conjugates. However, most of these reactions are not suitable for cross-linking with proteins due to the lack of complementary reactive groups in protein and/or due to the necessity for additional reagents or catalysts. Bioconjugation and cross-linking of ONs or DNA with proteins are typically based on reductive aminations7,22−24 or conjugate addition25 of lysine, disulfide formation from cysteine,26−28 or nonspecific diazirine photo-cross-linking.29,30 These reactions were used for covalent trapping of DNA− protein complexes for X-ray analysis or other characterization20−28 or for pull-down analysis of DNA-binding proteins.30 Recently, we have developed31 vinylsulfonamidemodified 2′-deoxyribonucleoside triphosphates (dNTPs) suit© 2016 American Chemical Society
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RESULTS AND DISCUSSION
Base-modified dNTPs are good substrates for DNA polymerases and suitable building blocks for enzymatic synthesis of modified ONs or DNA.39 Therefore, we designed dNTPs bearing the CA reactive group linked through a propargyl tether to position 5 of cytosine or to position 7 of 7-deazaadenine. Apart from the modified dNTPs, we also needed the corresponding nucleoside Received: June 24, 2016 Revised: July 28, 2016 Published: August 1, 2016 2089
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Scheme 2. Reaction of Cys or GSH with dCCAMP or dACAMP
monophosphates as stable model compounds for the testing of bioconjugations. Since the Sonogashira reaction of halogenated dNTPs with terminal acetylenes is a reliable single-step method for introduction of functionalized alkyne-linked substituents,31,39,40 we envisaged N-(propargyl)-chloroacetamide (1)41 as a suitable building block and prepared it from propargylamine and chloroacetyl chloride. The aqueous Sonogashira reactions of 5iodo-2′-deoxycytidine mono- (dC I MP) or triphosphate (dCITP), as well as of 7-iodo-7-deazaadenine dNMP (dAIMP) or dNTP (dAITP) with propargyl-chloroacetamide 1, were performed under previously reported conditions in the presence of Pd(OAc)2 and triphenylphosphine-3,3′,3″-trisulfonate (TPPTS) in a water/acetonitrile mixture (2:1).31 The reactions proceeded smoothly to furnish the desired chloroacetamidemodified nucleotides dCCAMP, dCCATP, dACAMP, and dACATP in moderate (but acceptable) yields (Scheme 1).
Scheme 3. Reaction of Histydyl Side Chain with dCCAMP Scheme 1. Synthesis of Chloroacetamide Linker and Desired Modified Mono- and Triphosphates
The modified nucleoside triphosphates dCCATP and dACATP were then tested as substrates for DNA polymerases in primer extension (PEX) experiments. The question was whether polymerases will be able to tolerate the presence of a reactive chloroacetamide group. Of the tested polymerases KOD XL performed the best, giving clean full-length ON products which were characterized by PAGE and MALDI (Figure 1A, Table 1; for sequences see Table S1). With the promising results in PEX in hand, we tested the triphosphates dCCATP and dACATP in the more challenging polymerase chain reaction (PCR), which requires not only that the modified dNTPs are good substrates for the polymerase, but also that the enzyme must be able to read through modified DNA template. The PCR reactions using Pwo, Vent(exo-), or KOD XL polymerase and either a 98-mer or 339-mer template worked well for dCCATP giving clean and strong bands of the modified dsDNA, but not for dACATP (Figure 1B; for sequences see Table S2 in Supporting Information). In order to test the cross-linking of modified ONs and short dsDNA, we prepared biotinylated dsDNA containing reactive CA-groups (30PEX1CCA) through PEX using biotinylated template and dCCATP as a substrate (Figure 2A). The modified DNA was then reacted with cysteine, Cys-containing undecapeptide (pept) in TEEA buffer pH 8.4, or histidine in phosphate buffer pH 7, followed by magnetoseparation of the template strand using streptavidine-coated magnetic beads42 (Figure 2A). The single-stranded ON products of cross-linking, 30ON1CCA_Cys and 30ON1CCA_pept, were successfully characterized by MALDI (Table 2). Formation of the product
Next, the modified monophosphates dCCAMP and dACAMP were used as model compounds in testing of cross-linking reactions with amino acids and peptides. First, we studied their reactions with 1.1 equiv of cysteine or glutathione (GSH) in triethylammonium acetate (TEEA) buffer (pH 8.4, to partly ionize SH group of Cys). In all cases we observed within 1 h a nearly quantitative formation of the products of nucleophilic displacement of chlorine by the thiol group of Cys or GSH. The nucleotide-Cys and -peptide conjugates dC CA MP_Cys, dACAMP_Cys, dCCAMP_GSH, and dACAMP_GSH were isolated in ca. 95% yield and fully characterized (Scheme 2). The chloroacetamide group appears to be more reactive compared to the previously reported VS-modified nucleotides,31,32 where the Michael addition required a higher excess of GSH (3 equiv) and longer reaction times to reach completion. Also unlike in the case of the thiol Michael adducts to VS-linked nucleotides,31,32 we have not observed any hydrolysis byproducts showing that this linkage is more stable. Next, we explored the potential for cross-linking with the imidazole group in the histidine side chain. We incubated the model reactive nucleotide dCCAMP with histidine (10 equiv) in phosphate buffer pH 7 at room temperature (Scheme 3). The reaction was sluggish and, after 1 week, an inseparable mixture of two isomeric adducts (in ratio 2:1) was formed in low yield of 28%. 2090
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Table 2. MALDI Data of Adducts of Modified ONs with Cysteine and Undecapeptide
Table 1. MALDI Data of Modified ONs ON
M (calcd) (Da)
M (found)[M + H] (Da)
6080.5 9333.7 6105.5
6081.6 9334.1 6106.0
M (calcd) (Da)
M (found) [M + H] (Da)
9419.4 10242.6
9420.6 10243.9
The DNA−protein cross-linking was studied with the GSTtagged core domain of tumor suppressor p53 (GSTp53CD) and its mutants.42,43 This protein contains two Cys residues (C275 and C277) which are in close proximity to bound DNA.44 The C277 participates in the recognition of the p53 consensus DNA sequence and thus is available for cross-linking, whereas the C275 points in the p53-DNA complex in the opposite direction and should not cross-link (as previously shown31,32 with VSmodified DNA). Therefore, we prepared mutants of the GSTp53CD with either C275 or C277 replaced by serine (to test specificity of the thiol-alkylation), a mutant with C277 replaced by histidine (to test whether also histidine can cross-link with the CA-probe), and a mutant R273H which loses specific recognition of the DNA sequence and did not cross-link with VSDNA in our previous study.31,32 Then, we prepared several DNA probes containing two modified nucleotides, either dCCA or dACA, in the sequence. The first two dsDNA probes contained dCCA or dACA in the consensus sequence (DNACCA and DNAACA). The other two probes contained the modified nucleotides in a mutated sequence with reversed order of C and A (CATG replaced by ACTG) DNArCCA and DNArACA (list of oligonucleotides used or synthesized for this study in Supporting Information Table S1). The ability of p53 protein and its mutants to recognize modified DNA was tested by incubation of the particular DNAprobes with proteins for 30 min on ice and the outcome was monitored by 5% EMSA (see Figure S8 in Supporting Information) which confirmed that p53 and its mutants (with the exception of the R273H mutant) recognize and (either noncovalently or covalently) bind to the modified DNA. The covalent cross-link formation was tested by incubation of the DNA with proteins for 2 h at r.t. followed by monitoring by denaturing SDS PAGE. The conversions of these cross-linking reactions were calculated from the relative intensity of the bands of the cross-linked conjugates versus total DNA using ImageJ Quantificator (Table 3). Figure 3A shows that both GST-tagged p53CD and p53CD gave covalently cross-linked products. The A-modified DNAACA (containing dACA) seemed to be more reactive and efficient (conversions of 50−60%) than DNACCA
Figure 1. (A) Primer extension using KOD XL polymerase and temp30_1C (lanes 2−4), temp30_2C (lanes 5−7), temp30_1A (lanes 8−10), or temp30_2A (lanes 11−13). Lane 1 (P): primer; lanes 2, 5, 8, 11 (+): natural dNTPs; lanes 3, 6, 9, 12 (C-/A-): negative control without dCTP or dATP; lanes 4, 7 (CCA): dCCATP, dTTP, dGTP, dATP; lanes 10, 13 (ACA): dACATP, dTTP, dGTP, dCTP. (B) Agarose gel analysis of PCR products amplified by Pwo, Vent(exo-), or KOD XL DNA polymerase; lane 1 (L): ladder; lanes 2, 7, 12 (+): natural dNTPs; lanes 3, 8, 13 (A−): dCTP, dGTP, dTTP; lanes 4, 9, 14 (C−): dATP, dTTP, dGTP; lanes 5, 10, 15 (ACA): dACATP, dCTP, dGTP, dTTP; lanes 6, 11, 16 (CCA): dCCATP, dATP, dTTP; conditions: 1.3% (2%) agarose gel for 339-mer (98-mer) stained with GelRed.
19ON_1CCA 30ON_1CCA 19ON_1ACA
ON 30ON1CCA_Cys 30ON1CCA_pept
Table 3. Conversions of Cross-Linking Reactions of Modified DNA with Proteinsa
Figure 2. (A) Enzymatic synthesis of chloroacetamide bearing DNA (DNACCA) and reaction with Cys and peptide followed by magnetoseparation. (B) Incorporation of dC CATP to oligonucleotide 30PEX1CCA and its conjugation with undecapeptide (pept). P: Primer; + natural dNTPs; C-: dTTP, dATP, dGTP; CCA: dCCATP, dTTP, dATP, dGTP; CCA+pept: incubation of the PEX product with peptide.
of cross-linking with undecapeptide was also visible on PAGE (Figure 2B). No reaction of 30PEX1CCA with histidine was observed within 1 week.
protein
DNAACA (%)
DNACCA (%)
p53CD GSTp53CD GSTp53_C275S GSTp53_C277S GSTp53_C277H GSTp53_R273H p53FLN268D GSTp63CD
51.4 ± 0.8 60.3 ± 0.9 26.3 ± 0.4 n.r.b 44.3 ± 1.0 14.9 ± 0.5 45.2 ± 3.2 46.9 ± 0.6
42.7 ± 0.8 45.5 ± 0.7 19.0 ± 0.1 n.r.b 16.2 ± 0.5 15.6 ± 0.5 29.3 ± 0.6 32.2 ± 3.6
a
Values are means of three independent experiments given with standard deviations. bNo reaction observed.
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in the core DNA binding domain (including the DNA contacting cysteine; lanes 9 and 18). The presence of GSTp53CD-DNA covalent cross-links has been also confirmed by Western blotting followed by immunodetection of p53 (Figure 3B). The gels show strong bands of free GSTp53CD at ca. 50 kDa and less mobile spots of cross-linked conjugates at ca. 60 kDa (lanes 5, 7, 9, and 11). Also here, it is clearly visible that CA-group is more reactive when attached to A than if attached to C. The DNA probes with mutated (reversed) recognition sequence DNArCCA and DNArACA gave essentially the same efficient cross-linking as the consensus sequences, which shows that the different reactivity of ACA compared to CCA is not caused by sequence. Since in model experiments with dNCAMPs (Scheme 2) we have not observed any significant differences in reactivity, the most likely explanation could be in the different orientation (and thus different steric hindrance) of the reactive group in the major groove of DNA either in 5-substituted cytosine or in 7substituted 7-deazaadenine.
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CONCLUSIONS We have developed chloroacetamide-linked nucleoside triphosphates dCCATP and dACATP which are good substrates for DNA polymerases and can be used for enzymatic synthesis of reactive DNA probes by primer extension. The dCCATP (but not dACATP) is also an efficient substrate for PCR. The modified nucleotides and ONs react readily with cysteine and Cyscontaining peptides through alkylation. The chloroacetamide moiety is apparently more reactive toward Cys than previously reported vinylsulfonamide.31,32 On the other hand, the reaction of model nucleotide dCCAMP with histidine was sluggish and inefficient whereas the corresponding CA-modified ONs did not react with His. CA-modified DNA probes were designed and prepared to study the DNA−protein cross-linking and were tested with p53 protein and several mutants. The DNA containing CAmodification in the recognition sequence efficiently cross-linked with p53 through alkylation of cysteine C277 and the dACA modification was more efficient than dCCA. The dNCA-modified DNA also reacted with histidine in a p53 mutant C277H. Unlike the previously reported Michael acceptor VS-modification,31,32 the CA-modified DNA probes also cross-linked with nonspecific R273H mutant showing that the higher reactivity of the CAgroups is at the expense of selectivity. Nevertheless, the chloroacetamide nucleotides and DNA probes seem to have a good potential in nucleic acid bioconjugation48 and in crosslinking with Cys- or His-containing proteins and will be further studied in pull-down assays and DNA-binding proteomics.
Figure 3. (A) Denaturing SDS PAGE analysis of cross-linking of modified DNA with different proteins (see the proteins below the gels, 1.3 equiv of protein to DNA) Lanes 1−9: DNAACA; lanes 10−18: DNACCA. Conditions: DNA (0.3 pM), protein 0.4 pM), pH 7.6, 0 °C/ 30 min then 25 °C/2 h. (B) SDS PAGE analysis of different DNAXCA_GSTp53CD conjugates followed by Western blot and protein immunnodetection; L: ladder.
containing dCCA (ca. 45%). The study of reactions with mutated proteins showed that the C275S mutant still gives the crosslinked product, though in lower conversions of 26% or 19%, respectively (lanes 4 and 13), whereas the C277S mutant does not (lanes 5 and 14). This indicates that the covalent cross-link was formed with cysteine C277 (in accord with our previous results31,32 with VS-modified DNA, Scheme 4). Interestingly, the Scheme 4. Covalent Cross-Linking of CA-Modified DNA with p53
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C277H mutant also gave cross-linked products with DNAACA (44%, lane 6) and with DNACCA (16%, lane 15). This suggests that histidine can also cross-link with CA-linked DNA if a proximity effect occurs. However, further studies with other proteins will be needed to fully characterize the histidine crosslinking. Surprisingly, the mutant R273H which did not show strong recognition of DNA on native gel also formed some weakly cross-linked products (ca. 15%, lanes 7 and 16). Successful cross-linking of dCCA- and dACA-modified DNA was also observed with full-length p53 (structure-stabilizing mutant p53FLN268D45,46 retaining the C277 residue and overall DNA binding properties of wt p53; lanes 8 and 17), as well as GSTp63CD47 derived from human p63 protein, a member of p53-family transcription factors sharing with p53 65% homology
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00342. Complete Experimental Section with procedures and characterization of all compounds and conjugates, additional gels, and copies of spectra (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. 2092
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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REFERENCES
This work was supported by the Academy of Sciences of the Czech Republic (RVO: 61388963, 68081707, and Praemium Academiae award for M. H.), by the Czech Science Foundation (P206-12-G151 to A. O., M. F., and M. H.), and by Gilead Sciences Inc.
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DOI: 10.1021/acs.bioconjchem.6b00342 Bioconjugate Chem. 2016, 27, 2089−2094
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DOI: 10.1021/acs.bioconjchem.6b00342 Bioconjugate Chem. 2016, 27, 2089−2094
