JULY/AUGUST 1998 Volume 9, Number 4 © Copyright 1998 by the American Chemical Society
COMMUNICATIONS A Method for the Oligomers
32P
Labeling of Peptides or Peptide Nucleic Acid
Igor A. Kozlov,† Peter E. Nielsen,‡ and Leslie E. Orgel*,† The Salk Institute for Biological Studies, P.O. Box 85800, San Diego, California 92186, and Center for Biomolecular Recognition, IMBG, The Panum Institute, Blegdamsvej 3c, DK-2100 N Copenhagen, Denmark. Received March 24, 1998
A novel approach to the radioactive labeling of peptides and PNA oligomers is described. It is based on the conjugation of a deoxynucleoside 3′-phosphate with the terminal amine of the substrate, followed by phosphorylation of the 5′-hydroxyl group of the nucleotide using T4 polynucleotide kinase and [γ-32P]ATP.
During the course of work on chimeras of DNA and peptide nucleic acids (PNAs), we needed to synthesize conjugates of 2′-deoxycytidine 3′-phosphate (dCp) and PNAs. Reaction between 2′-deoxycytidine 3′-phosphoroimidazolide (dCpIm) and the free terminal amino group of PNA was found to proceed with almost quantitative yield. We found that the conjugate could then be phosphorylated using T4 polynucleotide kinase (Maniatis et al., 1982) as efficiently as an oligonucleotide. This is not surprising since T4 polynucleotide kinase is known to phosphorylate nucleoside-containing substrates with a free 5′-OH group and an esterified phosphate group at the 3′-position (Richardson, 1981). Our experiments show that T4 polynucleotide kinase will accept a substrate in which the 3′-phosphomonoester is replaced by * Author to whom correspondence should be addressed. Telephone: (619) 453-4100, ext 1321. Fax: (619) 558-7359. E-mail:
[email protected]. † The Salk Institute for Biological Studies. ‡ Center for Biomolecular Recognition.
an analogous 3′-phosphoamide. We have now extended our procedure to the phosphorylation of other aminecontaining molecules and have used it to label the N terminus of a peptide. 2′-Deoxycytidine 3′-phosphoroimidazolide (dCpIm) was synthesized from dCp by an obvious modification of a published procedure in almost quantitative yield (Joyce et al., 1984). The PNA molecule H2N-ATGCTCTGCCONH2 (PNA-1) with a free terminal amino group and a protected carboxyl group was obtained by solid phase synthesis using a published procedure, and its identity was confirmed by mass spectroscopy (Christensen et al., 1995; Hyrup and Nielsen, 1996). The peptide molecule H2N-WQFEQQ-CONH2 (P-1) containing a free terminal amino group and a protected carboxyl group was synthesized at the Salk Institute for Biological Studies. To synthesize the PNA-dCp conjugate, a solution of PNA-1 (1 mM) and dCpIm (0.1 M) in 0.1 M Im buffer, at pH 7.7, was kept at room temperature for 2 days. The peptide-dCp conjugate was prepared in the same way
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Figure 1. (A) Scheme for the synthesis of a conjugate of PNA with dCp. (B) Scheme for the synthesis of a conjugate of a peptide with dCp: (i) 0.1 M Im buffer at pH 7.7 and (ii) T4 polynucleotide kinase and ATP, under routine conditions (Maniatis et al., 1982).
Figure 2. (A) Synthesis (a and b) and phosphorylation (c and d) of the conjugate between PNA-1 and dCp: (a) starting PNA-1, (b) reaction mixture after 2 days, (c) purified conjugate, (d) reaction mixture after phosphorylation of the conjugate, (1) starting PNA-1, (2) conjugate of PNA-1 with dCp and (3) 5′-phosphorylated conjugate. HPLC analysis was performed on an RPC5 column as previously described (Joyce et al., 1984; Schmidt et al., 1996). Reaction products were eluted with a linear gradient of NaClO4 (pH 12, 0 to 0.06 M over the course of 60 min) and monitored by UV absorption at 254 nm. (B) Synthesis (a and b) and phosphorylation (c and d) of the conjugate of peptide P-1 with dCp: (a) starting peptide, (b) reaction mixture after 2 days, (c) purified conjugate, (d) reaction mixture after phosphorylation of the conjugate, (1) starting peptide P-1, (2) conjugate of P-1 with dCp and (3) 5′-phosphorylated conjugate. HPLC analysis (a and b) was performed on a C18 RP-HPLC column using a linear gradient of 0 to 40% acetonitrile in water (0.1% TFA) over the course of 30 min. Reaction products were monitored by UV absorption at 220 nm. HPLC analysis (c and d) was performed on an RPC5 column as previously described (Joyce et al., 1984). Reaction products were eluted with a linear gradient of NaClO4 (pH 8, 0 to 0.06 M over the course of 60 min) and monitored by UV absorption at 220 nm. We did not study the nature of the material that eluted ahead of compound 2 in part c since the peak appears in a control experiment from which the substrate peptide was omitted. We believe that the large peak that elutes ahead of compounds 2 and 3 in part d corresponds to a component of the phosphorylation reaction mixture.
but using 3 mM peptide P-1 in place of 1 mM PNA. The reaction sequence is shown in Figure 1. The PNA-dCp conjugate was purified by HPLC on an RPC5 column at
pH 12 (Joyce et al., 1984; Schmidt et al., 1996) (Figure 2A, part b) and then dialyzed against water (Spectrum dialysis membranes). The peptide-dCp conjugate was
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on 20% polyacrylamide gels under standard conditions (Maniatis et al., 1982) (Figure 3). The three negative charges introduced by the terminal nucleotide are sufficient to transform a PNA or peptide molecule into a molecule suitable for electrophoretic analysis. The procedure described above should be applicable to the 32P labeling of biopolymers containing primary amino groups. dCp could be replaced by any other 2′-deoxynucleoside 3′-phosphate. Peptides that include lysyl residues are likely to undergo multiple conjugations. Some selectivity might be achieved by carrying out reactions at low pH. 32P labeling of biopolymers that lack amino groups should be possible using appropriate linkers if sulfhydryl or carboxyl groups are available. Other procedures for the 32P labeling of peptides and PNA oligomers have been described. They involve the attachment of short peptides that are substrates of protein kinases to the target molecules (Kemp and Pearson, 1991; Koch et al., 1995). The nature of the potential application will determine the method of choice. ACKNOWLEDGMENT
This work was supported by NASA Grant NAG5-4118 and NASA NSCORT/EXOBIOLOGY Grant NAG5-4546. We thank Aubrey R. Hill, Jr., for technical assistance, Anthony G. Craig for mass spectrometry, Jill Meisenhelder for the synthesis of the peptide, and Bernice Walker for manuscript preparation.
LITERATURE CITED Figure 3. Analysis of 32P-labeled conjugates between (A) PNA-1 and dCp and (B) peptide P-1 and dCp by electrophoresis on a 20% polyacrylamide gel. Separations were run for about 2 h at a constant current of 18 mA on a denaturing gel (8 M urea). The elution buffer was 50 mM Tris-borate (pH 8.3) containing 1 mM ethylenediaminetetraacetic acid (EDTA). Xc and Bpb are the positions of markers xylene cyanol and bromophenol blue, respectively.
purified by RP-HPLC on a C18 column (Figure 2B, part b). The fraction containing the product was neutralized with 1 N sodium hydroxide and then dialyzed against water. The molecular weights of the conjugates were determined using a Bruker Esquire-LC electrospray ionization mass spectrometer (PNA-dCp conjugate, calcd for C105H135N52O35P - H+ 2714, found 2715; peptide-dCp conjugate, calcd for C49H65H14O17P - H+ 1151.4, found 1150.9). The chemical structures of the conjugates were confirmed by their hydrolysis in 0.1 N HCl at 37 °C (2 h) which gave the initial PNA or peptide. These conditions are specific for the acid hydrolysis of phosphoamide linkages in nucleotide-peptide conjugates (Rjabova et al., 1965; Shabarova, 1970). The conjugates were phosphorylated using T4 polynucleotide kinase (Maniatis et al., 1982) (Figure 1). The reaction products were analyzed by HPLC on an RPC5 column at pH 12 for PNA (Figure 2A, part d) and pH 8 for peptide (Figure 2B, part d). Virtually complete phosphorylation of the PNA-dCp conjugate was achieved in 5 min, but complete phosphorylation of the peptidedCp conjugate required an initial 30 min of incubation with the enzyme followed by incubation for a further 30 min with a fresh portion of the enzyme. The 32P-labeled PNAs and peptides could be analyzed by electrophoresis
Christensen, L., Fitzpatrick, R., Gildea, B., Petersen, K. H., Hansen, H. F., Koch, T., Egholm, M., Buchardt, O., Nielsen, P. E., Coull, J., and Berg, R. (1995) Solid-phase synthesis of peptide nucleic acids. J. Pept. Sci. 3, 175-183. Hyrup, B., and Nielsen, P. E. (1996) Peptide nucleic acids (PNA): synthesis, properties and potential applications. Bioorg. Med. Chem. 4, 5-23. Joyce, G. F., Inoue, T., and Orgel, L. E. (1984) Non-enzymatic template-directed synthesis on RNA random copolymers: poly(C,U) templates. J. Mol. Biol. 176, 279-306. Kemp, B. E., and Pearson, R. B. (1991) Design and use of peptide substrates for protein kinases. Methods Enzymol. 200, 121133. Koch, T., Naesby, M., Wittung, P., Jorgensen, M., Larsson, C., Buchardt, O., Stanley, C. J., Norden, B., Nielsen, P. E., and Orum, H. (1995) PNA-peptide chimerae. Tetrahedron Lett. 36, 6933-6936. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Richardson, C. C. (1981) Bacteriophage T4 polynucleotide kinase. In The Enzymes (P. D. Boyer, Ed.) Academic Press, New York. Rjabova, T. S., Shabarova, Z. A., and Prokofiev, M. A. (1965) Selective cleveage of phosphoamide bond in adenylil-(5′-N)peptides. Dokl. Akad. Nauk SSSR 162, 1068-1070. Schmidt, J. G., Nielsen, P. E., and Orgel, L. E. (1996) Separation of “uncharged” oligodeoxynucleotide analogs by anion-exchange chromatography at high pH. Anal. Biochem. 235, 239-241. Shabarova, Z. A. (1970) Synthetic nucleotide-peptides. In Progress in Nucleic Acid Research and Molecular Biology, pp 145182, Academic Press, New York.
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