PNA-peptide chimerae

Tetrahedron Letters, Vol. 36, No. 38, pp. 6933-6936, 1995 Elsevier Science Ltd Printed in Great Britain 0040-4039195 $9.50+0.00

Pergamon 0040-4039(95)01373-3

PNA-Peptide Chimerae I Troels Koch*, Michael Naesby, Pernilla Wlttung ~, Mikkel J#rgensen, Charlotte Larsson, Ole Buehardt, Christopher J. Stanley, Bengt Nord4n n, Peter E. Nielsen#, and Henrik Orum*. PNA Diagnostics A/S, Lerse Parkall4 42, 2100 Copenhagen O. #Research Centre for Medical Blotechnology, Department of Biochemistry B, The Panum Institute, University of Copenhagen, 2100 Copenhagen O. r~Department of Physical Chemistry, Chalmers University of Technology, S-41296 Gothenburg, Sweden.

Abstract: Radioactivelabellingof PNA has been performedby linking a peptide segmentto the PNA which

is substmte for protein kinase A. The enzymaticphosphorylationproceeds in almost quantitativeyields. PNA (Peptide Nucleic Acid)(Scheme 1) is a synthetic DNA mimic which exhibits enhanced affinity and specificity for its complementary nucleic acid target sequence 28. The backbone of the PNA-oligomer is based on an oligo amide consisting of 2-aminoethyl-glycine units. Other uncharged amide based DNA mimics have also been proposed 9-tz, but PNA is the only example within this class of compounds to display outstanding DNA/RNA hybridization properties. Scheme I

R

R

o

HN

DNA:

PNA:

O I

O-P=O R' n

n

PNA oligomers are assembled using standard methods of peptide synthesis. This offers the intriguing possibility of forming PNA-peptide constructs (PNA-peptide chimerae) in a facile way. Such chimerae link the functionalities offered by peptides with the specific DNA and RNA recognition properties of PNA. In this letter we describe the synthesis and functional properties of the PNA-peptide chimera: H-Leu-Arg-ArgAla-Ser-Leu-GIy-Ado-Ado-Ado-TGT-ACG-TCA-CAA-CTA-Gly-NH z. The peptide segment ~3 (the "Kemptide") is a substrate for protein kinase A (PKA), which phosphorylates the serine residue. The chimera will be radioactively labelled if the phosphorylation is carried out with 32p phosphate. This makes PNA detectable by traditional biochemical tracer techniques. The chimera was synthesized manually by the improved solid phase PNA synthesis method L4which follows the Boc-strategy. The synthesis of both the peptide and PNA segments are fully compatible. The product was analysed by RP-HPLC and the target molecule represented 53% of the total integrated area in the crude sample. The identity was confirmed by electrospray mass spectroscopy (ESI; calculated/found: 5292.2/5291.0).

Function of PNA segment. To analyse whether the peptide segment influences the hybridisation properties of the PNA, the c h i m e r a (H-Leu-Arg-Arg-AIa-Ser-Leu-GIy-Ado-Ado-Ado-TGT-ACG-TCA-CAA-CTA-Gly-NH2), (Ado)fPNA (H-Ado-Ado-Ado-TGT-ACG-TCA-CAA-CTA-Gly-NHz) and "native" P N A (H-TGT-ACGTCA-CAA-CTA-GIy-NH2) were hybridized to oligonucleotides containing a complementary PNA target site. To detect a possible interaction between the peptide segment and the target DNA strand the oligonucleotides used were 40 mers carrying the PNA target site in the middle. As shown in Table 1 (row 1) the T m values increase slightly from native PNA to (Ado)3-PNA to unlabelled chimera. Part of this increase can be attributed to the 6933

6934

peptide segment of the chimera, where electrostatic interaction between the two positively charged arginines and the negatively charged phosphates of the DNA backbone is possible. Thus the T,, of the chimera decreases when it is phosphorylated as overall electroneutrality of the peptide segment is attained. IROW

U~abe~ Mismatch X=none X=Ado? (=Ado~-Kempfide

PNA/DNA COMPLEX

Labelled X =Ad%-Kemptide

1

5'*CFAGAGGATI]TAGTTGTGAC GTACAGGATC'FITrTCATAG-3' HzN-ATCAACAC'IGCATOT-X

[lOne

67.4°C 68.8°C

70.0oc

68.8°C

2

5'-OrAGAGGATCTAGqTGTGAAGTACAGGATCIWVFTCATAG.3 H~N-ATCAACAC3CrCATGTX

Gex^, Am ^

51.2°C 53.1°C

55.1°C

53.9°C

3

5'-CTAGAGGATCTAG'ITGTGATGTACAGGATCTITVfCATAG-3 H2N-ATCAACACqGCATGT-X

C~A/r~ ~

58.9°C 60.7°C

62.3°C

61.2°C

4

5'-CTAGAGG AFCTAGTI'GTGAGGTACAGGATCTYITI'CATAG3' H2N-ATCAACAC~ CATGT-X

G,~^/ Gr~^

53.1°C 53.8°C [

55.9°C

54.6°C

Table 1: Schematic representation of the T~ values of the PNA/DNA duplexes. Each hybridisation reaction contained 1.5 p.M PNA, 1.5 I.tM oligonucleotide, 10 mM Tris-HC1 (pH 8.0) and 100 mM NaCI.

The hybridisation specificity of the three PNAs were analysed by measuring the effect of introducing single base pair mismatches into the PNA/DNA duplexes. As shown in table 1 row 2-4 each mismatch gave almost similar T,, values with the three different PNAs. In summary, the Tr. measurements confirm that the affinity and specificity of the PNA segment of the chimera is essentially unaffected by the presence of the peptide domain. The structure of the duplex formed between the chimera and the 40 mer complementary oligonucleotide was analysed by circular dichroism (CD). The chimera alone gives essentially no CD spectrum, indicating that neither the peptide segment nor the PNA segment adopt any organised secondary structure (Fig.l). 15 10 5 0 -5

1

l

....

,mm., o l , ~ . , x l q ~ u .

-

n~IVe-PNA duplex

-

,o

272:2,: ©hlmera d~pkmx

-15

-20

200

220

240

260

280

300

320

Figure l : CD spectra of the chimera and the oligonucleotide alone and of the duplexes formed between the complementary oligonucleotide and the native PNA, (Ado)s-PNA and chimera. Experiments were performed in 5 mM phosphate buffer pH 7.0 at 20 °C.

Upon hybridisation with the complementary oligonucleotide very similar CD spectra were observed with all three PNAs (chimera, (Ado)~-PNA and native PNA), which all resembled that of the B-DNA duplex (PNA/DNA

6935

duplexes have previously been shown to adopt helical structures virtually similar to B-form DNA/DNA duplexesS). It is reasonable to conclude, therefore, that the peptide part of the chimera has no structural effect on the PNA-DNA duplex. In addition, one can exclude the possibility that the peptide forms a helical structure by interaction with the single stranded parts of the oligonucleotide flanking the target sequence. Function ofpeptide segment:. The chimera was labelled with 32P-),-ATP~5 which gave a specific activity of 2.4 x

106 cpm/pmol PNA. In the experiment was used 3ZP-y-ATP at a specific activity of 5000 Ci/mmol and the calculated maximum possible specific activity of the chimera would be 6 x 106 dpm/pmol. The enzymatic phosphorylation of the chimera was studied in a time course experiment (Fig. 2). 1 mU PKA was used to phosphorylate 1 nmol chimera. Samples were taken at different time points during the reaction and analysed on HPLC. Within 60 s ca. 20% of the chimera had been phosphorylated and more than 50% had been phosphorylated after 120 s. The reaction was completed within 300 s. The identity of the phosphorylated chimera was confirmed by mass spectroscopy (ESI; calculated/found: 5371.1/5370.9). In conclusion we have shown that peptides can be covalently linked to PNA molecules by a facile solid phase synthetic route. In the resulting PNA-peptide chimera the two segments are able to act independently of each other and exhibit highly specific interaction with their respective targets. This novel approach offers the possibility of creating a new class of synthetic multifunctional DNA-binding molecules. Further examples of PNA-peptide chimerae have been synthesised in our laboratory and their properties will be reported elsewhere.

mat

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Figure 2.

References and footnotes 1.

2.

3. 4.

5. 6.

* To whom correspondenceshould be addressed The term chimera designates a single molecularcomposition comprisingtwo structurally and functionally different segments. Abbreviationsand symbols used are standard oligo-peptide/nucleotide nomenclature:H-: deprotected terminal amino group; -NH2:C-terminal amido group; Boc: tert-butoxycarbonyl;HBTU: O-Benzotriazol-l-ylN,N,N",N'-TetramethyluroniumHexafluorophosphate;Ado: 8-amino-3,6-dioxaoctanoicacid. Nielsen, P.E.; Egholm, M.; Berg, R.H.; Buchardt, O. Science 1992, 254, 149%1500. Demidov, V.; Frank-Kalmenetskii, M.; Egholm, M.; Buchardt, O.; Nielsen, P.E. Nucl. Acid. Res. 1993, 21, 2103-2107. Buchardt, O.; Egholm, M.; Berg, R.H.; Nielsen, P.E. TIBS 1993, 1l, 384-386. Wittung, P.; Nielsen, P.E.; Buchardt, O.; Egholm, M.; Nord6n, B. Nature 1994, 368, 561-563. Nielsen, P.E.; Egholm, M.; Buchardt, O. Bioconjugate Chem. 1994, 5, 3-7.

6936

7.

8. 9.

10. 11. 12. 13. 14. 15.

Hanvey, J.C.; Peffer, N.J.; Bisi, J.E.; Thomson, S.A.; Cadilla, R.; Josey, J.A.; Ricca, J.D.; Hassman, C.F.; Bonham, M.AAu, .; G.K.; Carter, S.G.; Bruckenstein, D.A,; Boyd, A.L; Noble, S.A.; Babiss, L.E. Science 1992, 258, 1481-1485. Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier, S.M.; Driver, D.A.; Berg, R.H.; Kim, S.K.; Nord6n, B.; Nielsen, P.E. Nature 1993, 365, 566-568. Jones, S.A. Int. J. Biolog. Macromolecules 1979, 1, 194-207. De Koning, H.; Pandit, U.K. Rec. Tray. Chim. 1971, 91, 1069-1080. Garner, P.; Yoo, J.U. Tetrahedron Lett. 1993, 34, 1275-1278. Weller, D.D.; Daly, D.T.; Olson, W.K.; Summerton, J.E.J. Org. Chem. 1991, 56, 6000-6006. Pearson, B.P.; Kemp, B.E. Meth. Enzymol. 1991, 200, 62-8l Christensen, L.; Fitzpatrick, R.; Gildea, B.; Petersen, K.H.; Hansen, H.F.; Koch, T.; Egholm, M.; Buchardt, O.; Nielsen, P.E.; Coull, J.; Berg, R.H. submitted to J. Pept. Sci. 1994 (in press). 20 pmol of the chimera and (Ado)3-PNA (control PNA) was incubated separately in a reaction volume (50 gl) containing ~P-y-ATP (100 pmol >5000 Ci/mmol, Amersham), 50 mM MES (pH 6.9), 10 mM MgCI2, 0.5 mM EDTA, I mM DTT, 1 mg/mL BSA and 5 mU PKA (Boehringer Mannheim). After 30 rain at 30 °C the PNAs were separated from unincorporated ATP by ion exchange chromatography using diethylaminoethyl (DEAE) Sephadex A50 (Sigma) and the purified PNAs were counted in a scintillation counter. No radioactivity was found in the sample containing the control PNA.

(Received in UK 5 May 1995; revised 17 June 1995; accepted 2I July 1995)