Increased DNA binding and sequence discrimination of PNA oligomers containing 2,6-diaminopurine

Gerald Haaima 1 Henrik F. Hansen 1 2 Leif Christensen 0 1 Otto Dahl 1 Peter E. Nielsen 0 1 0 Department of Biochemistry and Genetics, Laboratory B, The Panum Institute , Blegdamsvej 3c, DK-2200 Copenhagen N, Denmark 1 Center for Biomolecular Recognition, Department of Chemistry, The H.C.rsted Institute, Universitetsparken 5 , DK-2100 Copenhagen , Denmark 2 Present address: PNA Diagnostics A/S, Rnnegade 2, DK 2100 Copenhagen , Denmark The synthesis of a diaminopurine PNA monomer, N-[N6-(benzyloxycarbonyl)-2,6-diaminopurine-9-yl] acetyl-N-(2-t-butyloxycarbonylaminoethyl)glycine, and the incorporation of this monomer into PNA oligomers are described. Substitution of adenine by diaminopurine in PNA oligomers increased the Tm of duplexes formed with complementary DNA, RNA or PNA by 2.5-6.5 C per diaminopurine. Furthermore, discrimination against mismatches facing the diaminopurine in the hybridizing oligomer is improved. Finally, a homopurine decamer PNA containing six diaminopurines is shown to form a (gel shift) stable strand displacement complex with a target in a 246 bp double-stranded DNA fragment. - Peptide nucleic acid (PNA) is a nucleic acid mimic in which the entire phosphodiester backbone has been replaced by a pseudopeptide (polyamide). This backbone consists of repeating units of aminoethylglycine with the nucleobases attached through a methylene carbonyl linker. It has been shown that PNA oligomers form highly stable complexes with complementary oligonucleotides (14). Mixed purine/pyrimidine oligomers of PNA form 1:1 complexes with WatsonCrick complementary DNA, RNA or PNA oligomers (4), whereas homopyrimidine PNA oligomers bind in a 2:1 (PNA:nucleic acid) mode (13,5). PNA has attracted broad attention in the fields of medicinal and bioorganic chemistry as well as in molecular biology and the chemistry and biological and physical properties of PNA were recently reviewed (69). 2,6-Diaminopurine (D) is a naturally occurring nucleobase found in, for example, the cyanophage S-2L, where it extensively replaces adenine (10). Since a diaminopurine-thymine base pair resembles a guanine-cytosine base pair (Fig. 1) in terms of both number of hydrogen bonds (three) and extent of interbase pair stacking overlap, duplex stabilization by D-T base pairs is expected. It has been shown that incorporation of diaminopurine into short DNA oligomers increases the thermal stability of the duplex by 02 C per D-T base pair (11). It has also been shown that the sequence specificity was improved in some cases (12). Thus a similar increased affinity and specificity upon incorporation of diaminopurine could be expected for PNAnucleic acid complexes. We report the synthesis of a diaminopurine monomer (N-[N6-(benzyloxycarbonyl)-2, 6-diaminopurine-9-yl] acetyl-N-(2t-butyloxycarbonylaminoethyl)glycine) and its incorporation into PNA oligomers. In addition, we show that incorporation of a D-T base pair into a PNANA heteroduplex increases the Tm by 2.56.5 C per AD substitution. Furthermore, we show that the sequence specificity can be increased upon incorporation of a diaminopurine nucleobase in PNA. Finally, it is demonstrated that a homopurine decamer PNA containing six diaminopurines (and four guanines) forms a strand displacement complex with a double-stranded DNA target and that this complex has sufficient stability to be analyzed by band shift assay. MATERIAL AND METHODS The A, C, G and T PNA monomers and N-(2-bocaminoethyl) glycine were purchased from Perseptive Biosystems. 2,6-Diaminopurine (98%) and standard chemicals were obtained from Aldrich. The phosphoramidites used for DNA synthesis were obtained from CruaChem and were incorporated into DNA oligomers according to standard protocols on a MilliGen/Biosearch 8700 DNA synthesizer. The N2,N6-diphenoxyacetyl-2,6-diaminopurine phos phoramidite was prepared according to the literature (11). RNA oligomers were purchased from DNA Technology and were used as received. Melting points are uncorrected. Flash chromatography was performed using Merck silica gel 60 (230400 mesh ASTM). 1H and 13C NMR spectra were obtained in DMSO-d6 using a Varian 400 MHz Unity or a Bruker 250 MHz AMX spectrometer. Chemical shifts were measured relative to solvent signals (DMSO-d6: d H = 2.50 p.p.m.; d C13 = 39.5 p.p.m.). FAB mass spectra were recorded on a Jeol Hx110/110 mass spectrometer operating in the positive ion mode. MALDI-TOF mass spectra of PNA oligomers were recorded on a Kratos Compact MALDI II instrument operating in the positive ion mode and using 3,5-dimethoxy-4-hydroxycinnamic acid as the matrix. HPLC was carried out on a 3.9 150 mm C-18 Delta Pak 5 m m 100 column (Waters). Eluant A: 99.9% H2O, 0.1% TFA; eluant B: 10% H2O, 89.9% CH3CN, 0.1% TFA; a linear gradient of 050% eluant B over 35 min at a flow rate of 1.0 ml/min monitored Scheme 1. (i) NaH, ethyl bromoacetate; (ii) N-benzyloxycarbonyl-N -methylimidazolium triflate; (iii) NaOH then HCl; (iv) DCC, DhbtOH, ethyl N-(2-bocaminoethyl)glycinate. at 260 nm. The column was heated to 50 C. The flow rate was 4.0 ml/min for preparative HPLC. The PNAs were synthesized according to a published protocol (13) except for one modification: following incorporation of the first 2,6-diaminopurine monomer the usual steps of capping unreacted amines were omitted. Syntheses typically gave a crude product of >90% purity as judged by reverse phase HPLC. The PNAs were purified by reverse phase HPLC and were characterized by MALDI-TOF mass spectrometry. All PNAs showed the expected mass within 5 m.u. (0.2%). Tm measurements Absorbance versus temperature was measured at 260 nm using a Gilford Response spectrophotometer. Heating was performed in 0.5 C steps from 5 to 90 C (~ 0.7 C/min). PNA oligomers were hybridized with complementary DNA sequences in a medium salt buffer containing 100 mM NaCl, 10 mM sodium phosphate and 0.1 mM EDTA, pH 7.0. The samples were heated to 90 C for 5 min, slowly cooled to 20 C and left at 4 C for at least 30 min prior to Tm measurements. The concentrations of all oligomers were determined using UV spectroscopy. The following molar extinction coefficients where used (l = 260 nm): A, 15.4 103; C, 7.3 103; G, 11.7 103; T, 8.8 103; 2,6-diaminopurine, 7.7 103 cm1M1. The 246 bp EcoRIPvuII fragment of a pUC19-derived plasmid containing a 5-GATCGAGAGGAAAA target cloned into the BamHI site was used for gel shift analyses and chemical probing. The plasmid DNA fragment was labeled with 32P ([a -32P]dATP/ Klenow DNA polymerase fragment) at the 3 EcoRI site. The 32P 3-end-labelled EcoRIPvuII fragment was incubated with the desired amount of PNA for 1 h in 100 m l 10 mM TrisHCl, 1 mM EDTA, pH 7.2, at room temperature (RT) and analyzed by gel electrophoresis in 5% polyacrylamide gels run in TBE (45 mM Trisborate, 1 mM EDTA, pH 8.5) buffer (gel shift) or by chemical probing with permanganate or diethylpyrocarbonate. Probing with diethylpyrocarbonate (5 m l) was performed for 5 min at R (...truncated)