Target DNA and PNA additives
For the preparation of ligand-modified pcPNAs, Boc-protected 2-thiouracil and 2,6-diaminopurine monomers were used with commercially available Boc-protected PNA monomers (from ASM), N
-phosphoserine (from Novabiochem) for control PNAs and (4-methylbenzhydryl)amine resin (from ABI) (please consult ref. 35
for detailed procedures of the preparation 2-thiouracil and 2,6-diaminopurine monomers and pcPNA synthesis). After the final removal of Boc or Fmoc group on pcPNA, the pcPNA strand was removed from the resin by “low-high TFMSA method.” All these modified and unmodified pcPNAs were purified by reversed-phase HPLC and characterized by MALDI-TOFMS (Bruker, AutoFLEX).
The 119-bp double-stranded DNA having the target sequence of synthesized pcPNAs was prepared by PCR and purified by QIAquick PCR Purification Kit (from Qiagen).
For the site-selective DNA scission, the target BFP plasmid DNA (4,733 bp) was prepared from pEGFP-N1 plasmid DNA (Clontech), which is coding closely-related green fluorescent protein (EGFP), by introducing mutations to give the corresponding BFP gene. The plasmid DNA was linearized by StuI and then used as a substrate for the site-selective DNA scission.
N2-/Fmoc/-N6-EDTP-2,6-diaminohexanoic acid (11)
The benzyl ester 10 (185.9 mg) was hydrogenated under an atmosphere of hydrogen over 10% Pd on carbon (61.9 mg) in methanol (15 ml) at room temperature for 2 h. The mixture was filtered, concentrated under reduced pressure to give the desired product as white powder, yield 151.0 mg (89%). 1H NMR (CDCl3, δ in ppm) 7.90 (s, 1 H, –CONHCH2–), 7.76 (m, 2 H, –ArH Fmoc), 7.62 (m, 2 H, –ArH Fmoc), 7.41–7.30 (m, 4 H, –ArH Fmoc), 5.73 (d, 1 H, J = 5.70 Hz, –NHCH–), 4.36–4.24 (m, 4 H, –NHCH– and –CH2CHAr Fmoc, overlapped), 4.00–3.92 (m, 1 H, –NCHCO–), 3.78–3.73 (m, 24 H, –OCH3), 3.41–3.16 (m, 12 H, –CONHCH2–, –CHCH2N– and –NCH2P–, overlapped), 1.99–1.32 (m, 6 H, –CHCH2CH2CH2–).
N2-Fmoc-N6-EDTA-2,6-diaminohexanoic acid (15)
The benzyl ester 14 (438.6 mg) was hydrogenated under an atmosphere of hydrogen over 10% Pd on carbon (72.6 mg) in methanol (10 ml) at room temperature for 3 h. The mixture was filtered, concentrated under reduced pressure to give the desired product as white powder, yield 325.4 mg (82%). 1H NMR (CDCl3, δ in ppm) 8.38 (m, 1 H, –CONHCH2–), 7.75 (m, 2 H, –ArH Fmoc), 7.60 (m, 2 H, –ArH Fmoc), 7.41–7.29 (m, 4 H, –ArH Fmoc), 5.65 (m, 1 H, –NHCH–), 4.36–4.22 (m, 4 H, –NHCH– and –CH2CHAr Fmoc, overlapped), 3.59–3.45 (m, 8 H, –NCH2P–), 3.40 (m, 2 H, –NCH2CH–), 3.33 (br s, 2 H, –NHCH2–), 2.95 (br s, 1 H, –NCHCO–), 1.89–1.32 [m, 42 H, –CHCH2CH2CH2– and –O(CH3)3, overlapped].
N,N-bis(methylenephosphonic acid tetrabenzyl ester) aminohexanoic acid (19)
The allyl ester 3 (508.0 mg) and tetrakis(triphenylphosphine)palladium(0) (83.4 mg) were dissolved in dichloromethane (10 ml) and stirred at 0°C. To the solution was added pyrrolidine (70 μl) and stirred at 0°C for 4 h. The reaction mixture was diluted with dichloromethane (20 ml) and washed with 1 N HCl aq (25 ml). The organic phase was dried over sodium sulfate, concentrated under reduced pressure and purified by silica column chromatography (eluting with 5% methanol in dichloromethane) to give the desired product as colorless oil, yield 426.9 mg (89%). 1H NMR (CDCl3, δ in ppm) 7.36–7.29 (m, 20 H, –ArH), 5.03–4.93 (m, 8 H, ArCH2-), 3.13 (d, 4 H, J = 8.4 Hz, –NCH2P–), 2.74 (t, 2 H, J = 6.95 Hz, εCH2), 2.24 (t, 2 H, J = 7.45 Hz, αCH2), 1.53 (tt, 2 H, both of J = 7.4 Hz, βCH2), 1.37 (m, 2 H, δCH2), 1.20 (m, 2 H, γCH2).
Gel shift assay to confirm the formation of invasion complex
Invasion complex composed of the 119-bp double-stranded DNA and pcPNAs with or without ligand was confirmed by gel-shift assay and its efficiency was evaluated. This 119-bp DNA was prepared from BFP plasmid DNA by PCR and the target sequence of pcPNAs is located in its middle. First, pcPNAs and target DNA were incubated in Hepes buffer (pH 7.0) at 50°C for 1 h. After the formation of invasion complex, loading buffer (0.05% bromophenol blue and 30% glycerol in 0.5 × TBE buffer) was added to the solution and then the mixture was subjected to 10% non-denaturing PAGE (PAGE). The bands were detected by staining with GelStar (from FMC). Invasion conditions are as follows; [double-stranded DNA (119 bp)] = 50 nM, [each of pcPNAs] = 200 or 400 nM and [Hepes (pH 7.0)] = 5 mM at 50°C for 1 h.
Evaluation of metal-chelating ability of ligand-modified PNA by the cerium-induced site-selective scission of double-stranded DNA
For the formation of invasion complex, linearized BFP plasmid DNA was incubated with pcPNAs in Hepes buffer (pH 7.0) at 50°C for 1 h. Then NaCl solution was added to the mixture and the site-selective DNA cleavage was started by adding Ce(NO3)3 solution to a final concentration. After a predetermined time, the scission reaction was stopped by adding an ethylenediamine-N,N,N′,N′-tetrakis(methylenephosphonic acid) (EDTP) 0.5 × TBE solution (pH 7.0) and the mixture was further incubated at 50°C for 1 h. After that, the scission products were subjected to 0.8% agarose gel electrophoresis and scission bands were detected by staining with GelStar. Typical cleavage conditions: [DNA] = 4 nM, [each of pcPNAs] = 100 nM, [Hepes (pH 7.0)] = 5mM, [NaCl] = 100mM and [Ce(NO3)3] = 30 μM at 50°C for 17 h under air.