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Artif DNA PNA XNA. Jul 1, 2011; 2(3): 79–89.
PMCID: PMC3324338

PNA HyBeacons for analysis of human mutations related to statin-induced myopathy

Abstract

Aminoalkyl and alkyne-tagged PNA HyBeacons have been synthesized, labeled with fluorescein via conventional amide bond or triazole formation (click chemistry) and used to detect single nucleotide polymorphisms (SNPs) implicated in statin-induced myopathy. The PNA HyBeacons gave much better mismatch/mutant discrimination than conventional DNA HyBeacons but smaller fluorescence changes on melting.

Keywords: fluorescence melting, genetic analysis, HyBeacon, PNA, SNPs, statin-induced myopathy

Introduction

Fluorogenic PCR1 using Taqman probes,2 Scorpions,3 and Molecular Beacons4 is an important technology in genetic analysis and diagnostics. Recent developments in high resolution melting analysis promise to revolutionise the field by adding an extra level of information,5 hence probe-based methods that are fully compatible with this technique are becoming increasingly important. HyBeacon probes6 (Fig. 1) are ideally suited for post-PCR melting analysis as their fluorescence emission changes greatly on duplex melting. Moreover, in the context of melting analysis, HyBeacons have advantages over other probe based methods as they are devoid of secondary structure.

Figure 1.
HyBeacon principle: In the unhybridized HyBeacon (A) the two fluorophores (black circles) interact and fluorescence is quenched. Binding to complementary target strand (B) leads to a highly fluorescent duplex (C).

Methods to increase the difference in thermodynamic stability between fully matched and mismatched probe-target duplexes7,8 are particularly valuable in forensic analysis, genetic testing and SNP detection by melting analysis. As PNA is known to give better mismatch discrimination than DNA,9 we decided to synthesize and evaluate PNA HyBeacons. A convenient synthesis of fluorescein PNA HyBeacons was developed using two labeling strategies (Fig. 2); click chemistry, the copper-catalyzed azide-alkyne cycloaddition (CuAAC reaction), and amide bond formation. Click chemistry10 has previously been used to label PNA,11 and is recognized for its high efficiency, whereas amide bond formation is a conventional technique for labeling amino-functionalised DNA12,13 and PNA. The major advantage of click chemistry over other labeling methods is the very high reactivity of alkynes with azides, and their lack of reactivity with almost all other functional groups.

Figure 2.
Modified PNA thymine monomers and PNA HyBeacon probes. 1, alkyne-T-PNA monomer; 2, aminoalkyl-T-PNA monomer; 3, 5-azidohexylfluorescein. Boxes: PNA HyBeacon containing triazole and amide bond linkages, FAM = fluorescein.

In this paper we describe an effective synthetic route to modified alkyne and amine-modified PNA monomers 1 and 2 and azidohexylfluorescein 3. In addition, we have evaluated PNA HyBeacons by probing single nucleotide polymorphisms (SNPs) at the Rs4149056 locus of the human genome that are correlated with statin-induced myopathy in high risk individuals.14

Results and Discussion

Synthesis of alkyne-T-PNA monomer 1 The synthesis of alkyne-T-PNA monomer 1 was accomplished according to Scheme 1. Synthesis of the PNA backbone 4 using 2,4-dinitrosulfonamide chemistry was efficient and straightforward.15,16 Alkylation of iodouracil with ethyl bromoacetate followed by Sonogashira coupling gave compound 6 in 75% overall yield. After hydrolysis of ester 6, carboxylic acid 7 was coupled to PNA backbone 4 to afford the fully protected PNA monomer 8 in 92% yield. Finally the tert-butyl ester of 8 was cleaved in acid to yield the monomer 1 in 78% yield (2:1 rotamers).

Scheme 1.
Synthesis of alkyne-T-PNA monomer 1: i) BrCH2CO2Et, K2CO3, DMF, 2 h, rt, 89%; ii) 1,7-octadiyne, CuI, Et3N, Pd(Ph3)4, DMF, rt, 2 h, 82%; iii) LiOH, 1,4 dioxane/H2O (2:1, v/v), rt, 30 min, Dowex 50 (H+), 82%; iv) HBTU, DIPEA, DMF, 3 h, 92%; v) 95:2.5:2.5, ...

Synthesis of aminoalkyl-T-PNA monomer 2 (Scheme 2). The modified base moiety of aminoalkyl-T-PNA monomer 2 was synthesized from propargylamine, iodouracil and 6-aminohexanoic acid. The first two steps were similar to alkyne-T-PNA monomer 1, briefly N-Boc-protected propargylamine 917 was coupled to modified iodouracil 5 via a Sonogashira coupling after which hydroysis of the ethyl ester under basic conditions gave the pure product in 82% yield over two steps. Compound 10 was coupled to PNA backbone 4 using PyBOP activator after which the Boc and tBu protecting groups of 11 were removed using trifluoroacetic acid to give compound 12. The free amino group of 12 was then coupled to N-Boc-aminohexanoic acid NHS ester 1318 to give the aminoalkyl-T-PNA monomer 2 in 63% yield (2:1 rotamers).

Scheme 2.
Synthesis of aminoalkyl-T-PNA monomer 2: i) Pd(PPh3)4, CuI, DMF, Et3N, 2 h, 90%; ii) LiOH, water/MeOH (1:3, v/v), 45 min, rt, then Dowex 50 (H+)/pyridine, 91%; iii) PyBOP, HOBt, 4, DIPEA, DMF, rt, 3 h, 63%; iv) 95:2.5:2.5, TFA:water:TIS (v/v), 30 min, ...

Synthesis of 5-azidohexylfluorescein 3. The synthesis of 5-azidohexylfluorescein 3 was achieved from the inexpensive, commercially-available precursors 6-aminohexanol and 5(6)-carboxyfluorescein in good yield (Scheme 3). 6-Aminohexanol was protected at the N-terminus using ethyl trifluoroacetate, followed by conversion of the alcohol to azide via tosyl derivative 15 to give compound 16. The trifluoroacetyl protecting group at the N-terminus was then removed with aqueous ammonia to give amine 17 which was coupled to the carboxyfluorescein dye 1819 using EDC chemistry giving, after purification, 5-azidohexylfluorescein 19 as a major product (24%) along with 6-azidohexylfluorescein (< 5%). Finally, the pivaloyl groups of compound 19 were removed under basic conditions using a mixture of ammonium hydroxide and 1,4-dioxane.

Scheme 3.
Synthesis of 5-azidohexylfluorescein 3 i) CF3CO2Et, DMAP, CH2Cl2 rt, 1 h; ii) tosyl chloride, Et3N, CH2Cl2, rt, 5 h, 70% for 2 steps; iii) K2CO3, DMF, NaN3, 50 °C, 5 h, 94%; iv) 35% NH4OH, 12 h, rt, then Dowex 50 (H+), 76%; v) EDC, HOBt, rt, 1 ...

PNA design and synthesis of PNA HyBeacon probes. To investigate the properties of PNA HyBeacons, a series of fluorescently labeled PNA probes for the human Rs4149056 target were synthesized using the following resource: (www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?rs=rs4149056) The wild-type probes (WP) contain thymine at the site of polymorphism and the mutant probes (MP) contain cytosine (Table 1).

Table 1.
PNA and DNA sequences, melting temperatures (Tms), quantum yields and MS analyses

Each PNA HyBeacon contains two or three fluorescein dyes with different spacings between them. Multiple fluorescent dyes are essential for HyBeacon to produce effective melting curves. PNA probes containing monomers 1 and 2 were synthesized by standard Fmoc solid-phase PNA synthesis methods and conjugation to the corresponding functionalised fluorescein PNAs was performed both in solution and on solid phases via click chemistry and amide bond formation.

In the click fluorescein labeling reaction under both solid and solution phase reaction conditions the ratio of alkyne PNA: 5-azidohexylfluorescein 3: copper sulfate: sodium ascorbate was 1:50:10:100 and in all cases the conversion was more than 80%. Dye-labeling via amide bond formation was performed by mixing 6-carboxyfluorescein succinimidyl ester with PNA containing free amino groups on aminoalkyl-T-PNA monomer 2 in a mixture of DMF: aqueous buffer (0.5 M NaHCO3/Na2CO3, 1:1, v/v, pH 8.75). The reactions were left at room temperature for 4–6 h and afforded the products in ~80% yield (calculated by RP-HPLC analysis). The purity of the crude PNA before and after labeling with three units of the modified PNA monomers was slightly lower than with two units. This is a consequence of less than quantitative coupling due to the bulk of fluorescein and the proximity of the modified bases. Example of RP-HPLC traces of PNA before and after labeling with fluorescein (amide bond formation) with three additions of the aminoalkyl-T-PNA monomer 2 and after click labeling with two additions of alkyne-T-PNA monomer 1 in solution phase are shown in Figure 3. The RP-HPLC of the crude PNA before labeling with the fluorescent dye shows the purity to be 70–80%. The product distribution after labeling in solution for three additions of the modified aminoalkyl-T-PNA monomer 2 with 6-carboxyfluorescein succinimidyl ester at positions 5, 9 and 12 (WP32Am) was 80% of the desired product and 15% of incomplete labeled products containing two fluorescein units. The click reaction to insert two additions of fluorescein at positions 5 and 12 went almost to completion (> 98%, WP6Cl2).

Figure 3.
RP-HPLC of PNA probes on C18 column A) Unpurified triply amine-labeled WP32U2, B) Unpurified triple fluorescein-labeled WP32Am, C) Unpurified doubly alkyne-labeled WP6U2, D) Unpurified doubly fluorescein-labeled WP6Cl2 where 1 = unlabeled PNA, 2 = incomplete ...

Fluorescence melting studies. Melting analyses (Table 1) were performed to evaluate the PNA HyBeacons using wild-type and mutant synthetic DNA targets (Wtt and Mtt) with adenine and guanine respectively at the site of polymorphism. Asymmetric PCR amplification and post-PCR melting analysis was also performed using a 10:1 ratio of primers RP:FP to produce excess of the target strand for the HyBeacon probes. These experiments were performed on a Roche LightCycler 1.5 and required a total of 60 min to complete the assay (50 min for PCR and 10 min for melting analysis). The fluorescein PNA HyBeacons with click triazole and amide linkages gave similar Tms (Fig. 4), but the melting peaks of the fluorescein amide PNA HyBeacons were slightly sharper than the triazole analogs. The Tms of mutant probes where the C base in the probe binds to mutant target (C:G base pair), were much higher (ΔTm = 17°C) than that of the wild-type target (C:A mismatch). For the wild-type probe with thymine at the point of discrimination (wild-type: target = T:A, mutant: target = T:G) the difference in Tm was 9°C. This is excellent discrimination considering the fact that TA is the least stable Watson-Crick base pair and GT is a relatively stable mismatch.

Figure 4.
Comparison of melting peaks of duplexes between PNA probes and asymmetric PCR products of Mtt and Wtt oligonucleotide targets. Probes were labeled by amide bond formation (Am) or click chemistry (Cl). WP = wild-type probe that contains a “T” ...

For effective SNP genotyping and mutation detection, the homozygote and heterozygote must be clearly distinguished. This was evaluated by mixing the Wtt and Mtt targets in the PCR reaction and analyzing the melting data. Examples of melting peaks of PNA probes hybridized to wild-type, mutant and mixed targets are shown in Figure 5. The discrimination between them is very clear. These curves represent the PNA probes bound to the Rs4149056 locus, the black curves show the homozygous alleles (TT and CC) and the red curves repesent the heterozygous allele (TC).

Figure 5.
Melting peaks of PNA HyBeacons with asymmetric PCR product of Mtt and Wtt oligonucleotide targets. Individual black melting peaks are homozygotes i.e., probe/wild-type target or probe/mutant target, melting peaks in red show the heterozygotes, i.e., probe ...

Comparing the ΔTms of the duplexes between HyBeacon probes with wild-type target and mutant targets, PNA HyBeacons probes gave much greater ΔTms than the DNA HyBeacon probe (up to 8°C difference). However, the fluorescence intensities of the hybridized PNA probes were lower than the DNA HyBeacon probes by up to a factor of four (Fig. 6). This is currently a limitation of PNA HyBeacons; the fluorescence change on duplex melting is rather small.

Figure 6.
Comparison of melting curves and melting peaks of DNA probe (MP DNA) and PNA probe (MP3Am) with their DNA targets (PCR products of Wtt and Mtt). Top and bottom graphs present melting curves and melting peaks of MP3Am (continuous line) and MP DNA (broken ...

Comparing the intensity of fluorescence of the DNA and PNA HyBeacons (Fig. 6), the duplex fluorescence is very similar whereas in the single strand the PNA is twice as fluorescent as the DNA. This indicates that the fluorophores in the single stranded PNA HyBeacon probe are not efficiently quenched. Quantum yields of the single stranded PNA and DNA probes and their duplexes with fully complementary synthetic oligonucleotide targets were determined and the results are shown in Table 1. The difference in quantum yield between the single and double stranded DNA probes was greater than for the equivalent PNA, which is in accordance with the fluorescence melting profiles. The melting curves of the PNA probes added before and after PCR amplification gave similar results, indicating that there was no inhibition of PCR by the PNA.

It is noteworthy that the ΔTms and melting peak resolution between the matched and mismatched duplexes are much greater for PNA than for DNA probes, a significant factor in clinical diagnostics. Other findings are: i) the order of stablility of the matched and mismatched PNA probes/targets duplex are C:G > T:A > T:G > C:A confirming that the stability trends for the PNA HyBeacons are similar to their DNA counterparts; ii) the mismatched PNA and DNA targets generated weaker fluorescence melting transitions than the fully matched systems. iii) the Tms of unlabelled PNA (U) were higher than the fluorescently labeled counterparts with an average ΔTm of 3.3–4.3°C per modified base; iv) the unlabelled PNA duplex with two and three additions of the aminoalkyl-T-PNA monomer 2 was more stable by 4°C than the unlabelled alkyne-T-PNA duplex containing monomer 1. This is probably due to the interaction of the positive charge of the protonated amino group and the phosphate backbone of the DNA target. However when the amine is conjugated to the fluorescent dye, the duplex is destabilized; v) placing two or three lysine residues at the carboxy-terminus of the PNA significantly improves solubility in aqueous media.

Conclusions

Fluorescein-labeled PNA HyBeacons have been synthesized by two methods; by click chemistry to create a triazole linkage, and by NHS ester-amine coupling for amide bond formation. In principle these approaches could be combined to offer orthogonality for labeling PNA with different dyes or other reporter groups at specific loci in the sequence. PNA HyBeacons give superior mismatch discrimination to DNA HyBeacons but much smaller fluorescence change on melting. The origin of the weaker fluorecence change is under investigation.

Materials and Methods

General

Reactions which required a dry atmosphere were conducted in flame-dried glassware under argon gas. Unless otherwise stated, commercial grade solvents and reagents were used without further purification. Tetrahydrofuran (THF) was distilled from sodium using benzophenone as an indicator. Triethylamine, pyridine, N,N diisopropylethylamine (DIPEA) and dichloromethane (DCM) were distilled from CaH2. Thin Layer Chromatography (TLC) which was performed on aluminum sheets coated with silica gel (0.22 mm thickness, aluminum backed, Merck) containing fluorescent indicator 60F254. Visualization was achieved by UV radiation at 254nm and in some cases an additional reagent, i.e., ninhydrin for amines, potassium permanganate for unsaturated compounds and Mary reagent for acids. Purification using column chromatography was performed with chromatography grade Silica gel 60A (particle size 35–70 μL and pore size 60 Å). NMR spectra were recorded using a Brüker AV 300 spectrometer or a Brüker DPX400 spectrometer. Proton-proton correlation (H-H COSY), carbon-proton correlation (C-H COSY) and DEPT-135 methods were also used to assign the spectra. Chemical shifts (δ) are given in ppm and J values are given in Hz and are correct to within 0.5 Hz. All spectra were internally referenced to the appropriate residual undeuterated solvent signal. The following abbreviations apply: “s” for singlet, “d” for doublet, “t” for triplet, “q” for quarter “br” for broad, “obsc” for obscure and “m” for multiplet. Low-resolution mass spectra (LRMS) were recorded using the electrospray technique on a Fisons VG platform instrument or a Waters ZMD quadrupole instrument. High-resolution mass spectra (HRMS) were recorded using the electrospray technique on a Bruker APEX III FT-ICR mass spectrometer.

ethyl (5-iodouracil-1-yl)acetate, 5 Potassium carbonate (6.28 g, 45.8 mmol) was added to a solution of 5-iodouracil (9.90 g, 41.6 mmol) in anhydrous N,N-dimethylformamide (80 mL) under argon, followed by slow addition of ethyl-bromoacetate (4.52 g, 40.8 mmol). The reaction mixture was stirred at room temperature and after 2 h, filtered through a pad of Celite, concentrated by co-evaporation with toluene (20 mL × 3) and purified by precipitation using dichloromethane and diethyl ether to yield the title product as an amorphous white solid (11.79 g, 89%). δΗ (300 MHz, CDCl3) 8.84 (1H, s, NH), 7.58 (1H, s, NCH), 4.48 (2H, s, NCH2), 4.31–4.24 (2H, q, J = 7.0 Hz, OCH2), 1.34–1.30 (3H, t, J = 7.0 Hz, CH3); δC (75.5 MHz, DMSO-d6) 167.0 (C), 160.1 (C), 150.2 (C), 148.6 (CH), 68.4 (C), 62.5 (CH2), 48.8 (CH2), 14.1 (CH3); LRMS-ESI+: m/z 324.9 [M+H]+(100%).

ethyl [5-(1,7-octadiynyl) uracil]acetate, 6 To a thoroughly degassed solution of ethyl (5-iodouracil-1-yl) acetate 5 (7.78 g, 24.0 mmol) in anhydrous N,N-dimethylformamide (100 mL) was added successively anhydrous triethylamine (10 mL), 1,7-octadiyne (3.82 mL, 36.0 mmol), tetrakis(triphenylphosphine)palladium(0) (2.80 g, 2.4 mmol) and copper (I) iodide (914 mg, 4.8 mmol). The mixture was left to stir under argon. After 2 h the reaction mixture was poured into a mixture of saturated ammonium chloride (75 mL) and EDTA (Na+ salt, 4.50 g in water 300 mL pH 8). The mixture was extracted with ethyl acetate (100 mL × 4) and the combined organic extracts were dried over sodium sulfate and filtered. The filtrate was concentrated in vacuo by co-evaporation with toluene (20 mL × 3) and the residue was purified by flash column chromatography eluting with 20% petroleum ether in dichloromethane graded to 2% methanol in dichloromethane, to give ethyl (5-octa-1,7-diynyl)acetate 6 as an amorphous white powder (5.94 g, 82%). δΗ (300 MHz, DMSO-d6) 11.7 (1H, s, NH), 7.98 (1H, s, NCH), 4.50 (2H, s, NCH2), 4.19–4.12 (2H, q, J = 7.0 Hz, OCH2), 2.77 (1H, t, J = 3 Hz, CCH), 2.40 (2H, m, CCCH2), 2.20 (2H, m, CH2CCH), 1.57 (4H, m, (CH2CH2CH2CCH), 1.20 (3H, t, J = 7.0 Hz, CH3); δC (75.5 MHz, DMSO-d6) 167.8 (C), 162.2 (C), 149.9 (C), 148.2 (CH), 98.2 (C), 92.9 (C), 84.2 (C), 72.5 (C), 71.3 (CH), 61.3 (CH2), 48.7 (CH2), 27.2 (CH2), 27.1 (CH2), 18.2 (CH2), 17.2 (CH2), 14.0 (CH3); LRMS-ESI+: m/z 342.5 [M+K]+(100%).

ethyl [5-(1,7-octadiynyl)-uracil]acetic acid, 7 A solution of compound 6 (1.00 g, 3.3 mmol) and lithium hydroxide (0.28 g, 6.6 mmol) in 2:1 1,4-dioxane:water mixture (9 mL) was stirred for 30 min at room temperature. The reaction was diluted with a 1:1 methanol:water (10 mL) and filtered through a pre-washed Dowex 50 (H+) column. The column was washed with methanol (20 mL) and the combined eluents were concentrated under reduced pressure. The residue was purified by column chromatography using dichloromethane graded to 20% methanol in dichloromethane, to provide the acid 7 as an amorphous white solid (0.74 g, 82%). δH (300 MHz, DMSO-d6) 13.19 (1H, s, COOH), 11.61 (1H, s, NH), 7.96 (1H, s, CCH2), 4.38 (2H, s, NCH2), 2.76 (1H, t, J = 3 Hz, CH2CCH), 2.39 (2H, m, CH2CCH), 2.19 (2H, m, CCCH2), 1.56 (4H, m, CH2CH2CH2CC); δC (75.5 MHz, DMSO-d6) 169.2 (C), 162.3 (C), 149.9 (C), 148.5 (CH), 97.9 (C), 92.8 (C), 84.2 (C), 72.6 (C), 71.3 (CH), 48.8 (CH2), 27.2 (CH2), 27.1 (CH2), 18.2 (CH2), 17.2 (CH2); LRMS-ESI-: m/z 273.2 [M-H]+(100%); HRMS calc. for C16H18N2NaO4 297.0851 found 297.0847.

bis-protected 5-(1,7-octadiynyl)uracil PNA monomer precursor, 8 A solution of the acid 7 (1.32 g, 4.8 mmol), HBTU (1.84 g, 4.8 mmol) and N,N-diisopropylamine (2.5 mL, 13.9 mmol) in N,N-dimethylformamide (6 mL) under argon was stirred at room temperature for 5 min before being added to a solution of compound 4 (2.0 g, 4.6 mmol) in N,N-dimethylformamide (6 mL) under argon. The reaction was stirred at room temperature for 3 h. The solvent was removed under vacuum. Ethyl acetate (200 mL) was added to the residue and the solution was washed with 10% citric acid (100 mL × 2) followed by saturated potassium chloride (100 mL × 1). The organic layer was dried over sodium sulfate and filtered. The filtrate was concentrated in vacuo and purified by flash column chromatography, eluting with dichloromethane graded to 2.5% methanol in dichloromethane. This afforded the title compound as a foam as a mixture of two rotamers (2.75 g, 92%). δH (300 MHz, CDCl3), (2:1 rotamers) 8.88 and 8.92 (3H, s, CONHCO), 7.96 (3H, s, NCH), 7.69 (6H, d, J = 7.4 Hz, ArH), 7.54 (8H, m, ArH and NH), 7.28 (12H, m, ArH), 5.95 and 5.43 (3H, t, J = 7.0 Hz, NHFmoc), 4.42–4.13 (15H, m, OCH2CH and CH2COOtBu), 4.00 and 3.89 (6H, s, NCH2CO), 3.52–3.28 (12H, m, NCH2CH2NHFmoc), 2.34- 2.30 (6H, m, CCCH2), 2.10–2.18 (6H, m, CH2CCH), 1.90–1.87 (3H, m, CCH), 1.60–1.55 (12H, m, CH2CH2CH2CCH), 1.44 and 1.40 (27H, s, CH3); δC (75.5 MHz, CDCl3): 168.7 (C), 162.8 (C), 162.1 (C), 156.7 (C), 149.9 (C), 147.2 (CH), 143.9 (C), 143.8 (C), 141.3 (C), 127.8 (CH), 127.1 (CH), 125.1 (CH), 120.0 (CH), 100.6 (C), 94.7 (C), 71.2 (C), 68.6 (C), 66.7 (CH2), 51.1 (CH2), 49.8 (CH2), 48.9 (CH2), 48.0 (CH2), 47.3 (CH), 39.2 (CH), 28.0 (CH3), 27.5 (CH2), 27.4 (CH2), 19.1 (CH2), 17.9 (CH2).; LRMS-ESI+: m/z 653.3 [M+H]+(100%).

alkyne-T-PNA monomer, 1 A solution of compound 8 (3.00 g, 4.6 mmol) in trifluoroacetic acid (9.5 mL), triisopropylsilane (0.25 mL) and water (0.25 mL) was stirred at room temperature for 30 min. The mixture was then added slowly to cold diethyl ether (100 mL) and the resultant solid was filtered and dried overnight under high vacuum to give the product as an amorphous white solid as two rotamers (2.14 g, 78%). δH (300 MHz, DMSO-d6), (2:1 rotamers) 12.96 (3H, br.s, COOH), 11.59 (3H, br.s, CONHCO), 7.88 (6H, d, J = 7.5 Hz, ArH), 7.80 (3H, s, NCH), 7.73 and 7.69 (3H, br.s, CONH), 7.68 (6H, d, J = 7.4 Hz, ArH), 7.41 (6H, dd, J = 7.5, 7.1 Hz, ArH), 7.35 (6H, dd, J = 7.5 Hz, 7.1 Hz, ArH), 4.71 and 4.53 (6H, s, CH2COOH), 4.34–4.23 (9H, m, CH2CHFmocH), 4.14 and 3.98 (6H, s, NCH2CO), 3.41–3.11 (12H, m, NCH2CH2NH), 2.75 (3H, m, CCH), 2.34 (6H, m, CCCH2), 2.16 (6H, br.m, CH2CCH), 1.53 (12H, br.m, CH2CH2CH2CCH); δC (75.5 MHz, DMSO-d6) 170.4 (C), 166.9 (C), 162.3 (C), 156.3 (C), 150.0 (CH), 148.7 (CH), 143.9 (C), 140.7 (C), 127.6 (CH), 127.1 (CH), 125.1 (CH), 120.1 (CH), 97.9 (C), 92.8 (C), 84.2 (C), 72.7 (C), 71.4 (C), 65.5 (CH2), 47.9 (CH2), 47.8 (CH2), 46.8 (CH2), 46.7 (CH), 40.1 (CH2), 39.0 (CH), 27.2 (CH2), 27.1 (CH2), 18.2 (CH2), 17.2 (CH2).; LRMS-ESI-: m/z 595.3 [M-H]-(100). HRMS [M+Na]+: m/z 619.2171 ; calcd. for C33H32N4NaO7 619.2168.

5-[(tert-butoxycarbonylamino)prop-1-ynyl]uracil acetic acid, 10 To a thoroughly degassed solution of ethyl (5-iodouracil-1-yl)acetate 5 (1.99 g, 6.1 mmol) in anhydrous N,N-dimethylformamide (5 mL) under argon was added successively anhydrous triethylamine (7 mL), tert-butyl prop-2-ynylcarbamate 9 (1.66 g, 10.7 mmol), tetrakis(triphenylphosphine)palladium(0) (0.63 g, 0.5 mmol) and copper (I) iodide (0.21 g, 1.1 mmol). The mixture was left to stir at room temperature. After 2 h the reaction was poured onto a mixture of saturated ammonium chloride (80 mL), aq. EDTA sodium salt (1.2 g in 80 mL water pH 8) and extracted with ethyl acetate (50 mL × 4). The organic fractions were combined and dried over sodium sulfate, filtered and the filtrate was concentrated in vacuo. The residue was co-evaporated with toluene (20 mL × 3), concentrated and purified by flash column chromatography eluting with 10% ethyl acetate in dichloromethane, gradually increasing to 80% ethyl acetate to give the ethyl ester of compound 10 as an amorphous off white powder (1.90 g, 90%). δH (300 MHz, DMSO-d6) 11.74 (1H, s, NHCO), 8.04 (1H, s, NCH), 7.33 (1H, t, J = 5.7 Hz, NHBoc), 4.50 (2H, s, CH2COOEt), 4.15 (2H, q, J = 7.2 Hz, CH2CH3), 3.92 (2H, d, J = 5.7 Hz, CH2NHBoc), 1.39 (9H, s, CH3), 1.20 (3H, t, J = 7.2 Hz, CH2CH3); δC (75.5 MHz, DMSO-d6) 167.8 (C), 162.0 (C), 155.2 (C), 149.9 (CH), 149.1 (C), 97.4 (C), 90.3 (C), 78.2 (C), 73.4 (C), 61.3 (CH2), 48.7 (CH2), 30.0 (CH2), 28.1 (CH3), 14.0 (CH3); LRMS-ESI+: m/z 352.4 [M+1]+(100). A solution of the ethyl ester (1.93 g, 5.5 mmol) and lithium hydroxide (0.46 g, 10.9 mmol) in methanol:water (3:1, 24 mL) was stirred at room temperature for 45 min. The reaction was filtered through Dowex 50 H+ [prewashed with 3 volumes of the following solvents: methanol, water, HCl (1 N), water, 50% pyridine in water and water] and the Dowex was washed with methanol/water (1:1 v/v, 100 mL) to fully recover the product. The eluents were combined and concentrated under high vacuum. The resultant yellow solid was washed with diethyl ether until it became white after which it was dried for 12 h under vacuum to give compound 10 as an amorphous white solid (1.61 g, 91%). δH (300 MHz, DMSO-d6) 13.23 (1H, br.s, COOH), 11.69 (1H, s, CONHCO), 8.03 (1H, s, NCH), 7.32 (1H, br.t, NHBOC), 4.42 (2H, s, CH2COOH), 3.92 (2H, d, J = 4.8 Hz, CH2NHBoc), 1.37 (9H, s, CH3); δC (75.5 MHz, DMSO-d6) 169.2 (2C), 162.1 (C), 155.2 (C), 149.9 (CH), 149.3 (C), 97.2 (C), 90.2 (C), 78.2 (C), 73.6 (C), 61.3 (CH2), 48.7 (CH2), 48.6 (CH2), 30.0 (CH2), 28.1 (CH3); LRMS-ESI+: m/z 324.5 [M+1]+(100); HRMS [M+Na]+: m/z 346.1013 ; calcd. for C14H17N3O6Na 346.1010.

fully protected 5-(aminoprop-1-ynyl)uracil PNA monomer precursor, 11 A solution of the compound 10 (1.39 g, 4.3 mmol), PyBOP (2.46 g, 4.7 mmol), HOBt (0.06 g, 0.4 mmol) and N,N-diisopropylamine (2.2 mL, 12.9 mmol) in N,N-dimethylformamide (6 mL) was stirred under argon at room temperature for 5 min before being added to a solution of PNA backbone 7 (1.67 g, 3.8 mmol) in N,N-dimethylformamide (6 mL) under argon. The reaction was stirred at room temperature for 3 h after which the solvent was removed under vacuum. Ethyl acetate (150 mL) was added and the resultant solution was washed with 10% citric acid (50 mL × 2) and saturated potassium chloride (50 mL × 2). The organic layer was dried over sodium sulfate, filtered and the filtrate was concentrated in vacuo before being purified by flash column chromatography using 20% ethyl acetate in dichloromethane, gradually increase polarity to 50% ethyl acetate to give compound 11 as an amorphous white powder (two rotamers). (1.711 g, 63%). δH (300 MHz, CDCl3), (2:1 rotamers) 7.75 (9H, d, J = 7.4 Hz, 6ArH and 3NCH), 7.62–7.56 (8H, d, J = 7.4 Hz, 6ArH and 2CONHCO), 7.43–7.28 (16H, m, 12ArH, 3NHBoc and CONHCO), 6.08 and 5.61 (3H, br.t, NHFmoc), 5.01&4.47 (4H, br.s, CH2COOtBu), 4.47–4.43&4.34 (11H, m, 3CH2CH and 2CH2COOtBu), 4.21&4.07 (6H, m, CH2NHBoc), 4.10 and 3.95 (6H, s, CHNCH2), 3.57–3.35 (12H, m, CH2CH2NHFmoc); δC (75.5 MHz, CDCl3) 187.3 (C),162.1 (C), 156.7 (C), 155.4 (C), 148.6 (CH), 143.8 (C), 141.2 (C), 127.8 (CH), 127.1 (CH), 126.3 (CH), 125.2 (CH), 120.1 (CH), 99.2 (C), 90.5 (C), 83.8 (C), 82.7 (C), 80.5 (C), 73.7 (C), 66.7 (CH2), 51.1 (CH2), 49.8 (CH2), 48.8 (CH2), 48.2 (CH2), 47.2 (CH), 39.2 (CH2), 31.2 (CH2), 28.4 (CH3), 28.0 (CH3); LRMS-ESI+: m/z 702.1 [M+H]+(100).

Fmoc-protected 5-(aminoprop-1-ynyl)uracil PNA monomer precursor, 12 A solution of compound 11 (1.09 g, 1.5 mmol) in trifluoroacetic acid (4.75 mL), triethylsilane (0.13 mL) and water (0.13 mL) was stirred at room temperature for 30 min. The resultant mixture was added slowly to cold diethyl ether (100 mL) and the solid was filtered and dried overnight under high vacuum to give the product as an amorphous white solid (two rotamers) (0.80 g, 95%). δH (300 MHz, CDCl3), (2:1 rotamers) 6.30 (9H, d, J = 8 Hz, ArH and NCH), 6.16 (6H, d, J = 7 Hz, ArH), 5.93–5.80 (12H, m, ArH), 3.38 and 3.14 (6H, s, CH2COOH), 2.92 and 2.77 (6H, d, J = 6.8 Hz, CH2CH), 2.75 and 2.63 (3H, br.t, CH2CH), 2.72 &2.44 (6H, s, CHNCH2), 2.47–2.02 (6H, m, CH2NH2), 2.03–1.83 (12H, m, CH2CH2NHFmoc); δC (75.5 MHz, CDCl3) 172.3 (C),169.3 (C), 151.8 (CH), 145.3 (C), 142.6 (C), 128.8 (CH), 128.2 (CH), 126.2 (CH), 121.0 (CH), 98.2 (C), 85.7 (C), 79.6 (C), 67.9 (CH2), 49.9 (CH2), 49.3 (CH2), 49.0 (CH2), 48.5 (CH), 39.9 (CH2), 30.8 (CH2); LRMS-ESI+: m/z 546.1 [M+H]+(95), 568.2 [M+Na]+(100); HRMS [M+Na]+: m/z 568.1802 ; calcd. for C28H27N5O7Na 568.1803.

aminoalkyl-T-PNA monomer 2 NHS ester 13 was added to a solution of compound 12 (0.34 g, 0.6 mmol) in N,N-dimethylformamide (2 mL) and N,N-diisopropylethylamine (0.32 mL, 1.8 mmol), and the reaction mixture was stirred at room temperature for 4 h. The solvent was concentrated in vacuo, and the product was purified by flash column chromatography using dichloromethane, gradually increase the polarity to 20% methanol in dichloromethane to give the pure compound as amorphous off white powder (rotamers) (0.29 g, 63%). δH (300 MHz, DMSO-d6), (2:1 rotamers) 11.62 (3H, br.s, COOH), 8.28 (3H, t, J = 5.0 Hz, CCCH2NH), 7.88 (6H, d, J = 7.4 Hz, ArH), 7.85 (3H, s, CONHCO), 7.67 (6H, d, J = 7.2 Hz, ArH), 7.58 (3H, br.t, NHFmoc), 7.41 (6H, dd, J = 7.2, 7.5 Hz, ArH), 7.32 (6H, dd, J = 7.4, 7.5 Hz, ArH), 6.75 (3H, t, J = 6.5 Hz, NHBoc), 4.69 and 4.54 (6H, s, CH2COOH), 4.32 - 4.20 (9H, m, CH2CH), 4.03 (6H, m, CCCH2NH), 4.86 and 3.80 (6H, s, CHNCH2), 3.38–3.13 (9H, m, CH2CH2NHFmoc), 2.87 (6H, m, CH2NHBoc), 2.79–2.73 (3H, m, CH2CH2NHFmoc), 2.06 (6H, t, J = 7.5 Hz, CH2(CH2)4NHBoc), 1.41–1.51 (6H, m, CH2(CH2)3NHBoc), 1.36 (27H, s, CH3), 1.36–1.29 (6H, m, CH2CH2NHBoc), 1.23–1.15 (6H, m, CH2(CH2)2NHBoc); δC (100.6 MHz, DMSO-d6) 172.2 (C), 167.8 (C), 162.6 (C), 156.8 (C), 156.0 (C), 150.5 (C), 144.4 (C), 141.2 (C), 128.0 (CH), 127.6 (CH), 125.7 (CH), 120.5 (CH), 97.4 (C), 90.1 (C), 77.7 (C), 74.5 (C), 65.9 (CH2), 49.1 (CH), 48.5 (CH2), 48.2 (CH2), 47.2 (CH2), 38.4 (CH2), 35.5 (CH2), 32.6 (CH2), 29.7 (CH2), 28.9 (CH2), 28.7 (CH3), 26.5 (CH2), 25.3 (CH2) ; LRMS-ESI-: m/z 757.2 [M-H]-(100). HRMS [M+Na]+: m/z 781.3160; calcd. for C39H46N6NaO10 781.3168.

6-(2,2,2-trifluoroacetamido)hexyl 4-methylbenzensulfonate, 15 To a mixture of 6-amino-1-hexanol (5.00 g, 42.7 mmol) was added dimethylaminopyridine (5.20 g, 42.7 mmol) in dichloromethane (215 mL) and ethyl trifluoroacetate (10.2 mL, 85.4 mmol). The reaction mixture was stirred at room temperature and after 1 h was concentrated under vacuum and co-evaporated with toluene (60 mL × 3) to give compound 14 which was dissolved in dichloromethane (215 mL). The mixture was cooled down to 0°C and triethylamine (11.9 mL, 85.4 mmol) and tosyl chloride (16.30 g, 85.4 mmol) were slowly added. The reaction was left to warm up to room temperature and was stirred for a further 5 h. It was then poured onto ice, washed with water (60 mL) and sodium hydrogen carbonate (60 mL × 3), dried over sodium sulfate, concentrated and purified by flash column chromatography, eluting with toluene in hexane (0–100%) to give 6-(2,2,2-trifluoroacetamido)hexyl-4-methylbenzensulfonate 15 as an amorphous solid (11.00 g, 70%). δH (400 MHz, DMSO-d6) 9.41 (1H, s, NH), 7.48 (2H, d, J = 7.5 Hz, ArH), 7.12 (2H, d, J = 7.5 Hz, ArH), 4.25 (2H, t, J = 6.5 Hz, CH2OS), 3.22–3.10 (2H, m, CH2NH), 2.29 (3H, s, CH3), 1.69–1.59 (2H, m, CH2CH2OS), 1.54–1.16 (6H, m, CH2CH2CH2CH2NH); δC (100.6 MHz, DMSO-d6) 155.5 (C), 145.5 (C), 137.7 (C), 128.0 (2CH), 125.4 (2CH), 76.0 (CH2), 38.9 (CH2), 28.9 (CH2), 28.0 (CH2), 25.5 (CH2), 24.2 (CH2), 20.7 (CH3); 19F NMR (DMSO-d6): d ppm -74.35 (CF3CO-); MS-ESI+: m/z 390.2 [M-H]-.

N-(6-azidohexyl)-2,2,2-trifluoroacetamide, 16 To a mixture of 6-(2,2,2-trifluoroacetamido)hexyl-4-methylbenzensulfonate 15 (5.60 g, 15.2 mmol) in anhydrous N,N-dimethylformamide (40 mL) was added potassium carbonate (3.20 g, 22.8 mmol) and sodium azide (2.0 g, 30.4 mmol). The mixture was stirred at 50°C for 5 h, filtered through a pad of celite and the solvent was removed in vacuo. The residue was co-evaporated with toluene (20 mL × 2), concentrated and purified by flash column chromatography, eluting with toluene in hexane (0–100%) to give N-(6-azidohexyl)-2,2,2-trifluoroacetamide 16 as a yellow oil (3.40 g, 94%). δH (400 MHz, DMSO-d6) 9.36 (1H, s, NH), 3.31 (2H, t, J = 6.8 Hz, CH2N3), 3.21–3.13 (2H, m, CH2NH), 1.57–1.44 (4H, m, CH2CH2NH and CH2CH2N3), 1.36–1.22 (4H, m, CH2CH2(CH2)2N3); δC (400 MHz, DMSO-d6) 155.0 (C),114.8 (C), 50.4 (CH2), 39.0 (CH2), 28.0 (CH2), 27.9 (CH2), 25.57 (CH2), 25.52 (CH2); δF (DMSO-d6) -74.35 (CF3CO-); MS-ESI+: m/z 237.2 [M+H]+; HR-ES [M+Na]+: m/z 261.0937 ; calcd. for C8H13F3N4NaO 261.09392.

6-azido-1-aminohexane, 17N-(6-azidohexyl)-2,2,2-trifluoroacetamide 16 (3.30 g, 14.0 mmol) was dissolved in aqueous ammonia solution (35%, 500 mL) and the reaction mixture was stirred for 12 h at room temperature. It was then concentrated to dryness, co-evaporated with methanol (30 mL × 2), redissolved in aqueous methanol (50:50) and passed through a column of Dowex H+ form 50W (500 mL) eluting with 3% aqueous ammonia in methanol (1:1). The eluent was collected, the solvent was evaporated and the product was dried under vaccuum overnight to give 6-azido-1-aminohexane 17 as a yellow oil (1.50 g, 76%). δH (400 MHz, DMSO-d6) 7.77 (s, 2H, NH2), 3.37–3.30 (2H, obsc by residue peak of DMSO, CH2CH2N3), 2.81–2.71 (2H, t, J = 7.5 Hz, CH2NH2), 1.59–1.46 (4H, m, CH2(CH2)2CH2CH2N3), 1.35–1.28 (4H, m, CH2CH2(CH2)2N3); δC (400 MHz, DMSO-d6) 50.4 (CH2), 38.8 (CH2), 27.9 (CH2), 26.7 (CH2), 25.5 (CH2), 25.2 (CH2); MS-ESI+: m/z 143.2 [M+H]+; HR-ES [M+H]+: m/z 143.1293; calcd. for C6H15N4 143.12967.

di-O-pivaloyl-6-[fluorescein-5-carboxamido]azidohexane, 19 To a solution of di-O-pivaloyl-5(6)-carboxyfluorescein 18 (0.66 g, 1.2 mmol) in anhydrous dichloromethane (12 mL) was added HOBt (0.32 g, 2.4 mmol) and EDC (0.46 g, 2.4 mmol). The reaction mixture was stirred under argon for 5 min, then 6-azido-1-aminohexane 17 (0.17 g, 1.2 mmol) was added. After 1 h the reaction mixture was concentrated and purified by flash column chromatography, eluting with ethyl acetate in hexane (0–40%) to give di-O-pivaloyl-6-[fluorescein-5-carboxamido]azidohexane 19 as a white foam (190 mg, 24%). A small quantity of the impure 6-isomer was also isolated. δH (400 MHz, CDCl3) 8.46 (1H, br. s, ArH), 8.23 (1H, d, J = 8.0 Hz, ArH), 7.26 (1H, d, J = 8.0 Hz, ArH), 7.07 (2H, br. s, ArH), 6.78 (5H, br. s, ArH and NH), 3.48 (2H, t, J = 6.7 Hz, CH2NH), 3.23 (2H, m, CH2N3), 1.70–1.53 (4H, m, 4H, CH2(CH2)2CH2 CH2NH), 1.45–1.37 (4H, m, CH2CH2(CH2)2NH), 1.35 (18H, s, CH3); δC (100.6 MHz, CDCl3) 176.4 (2C), 168.6 (C), 165.4 (C), 155.1 (C), 152.8 (C), 151.6 (C), 137.1 (C), 135.1 (CH), 128.7 (2CH), 126.4 (C), 124.5 (CH), 123.1 (CH), 117.8 (2CH), 115.5 (2C), 110.4 (2CH), 82.3 (C), 51.3 (CH2), 40.2 (CH2), 39.2 (2C), 29.3 (CH2), 28.7 (CH2), 27.0 (CH3), 26.4 (CH2), 26.3 (CH2); MS-ESI+: m/z 691.8 [M+Na]+; HR-ES [M+Na]+: m/z 691.2753 ; calcd. for C37H40N4NaO8 691.27438.

5-Azidohexylfluorescein 3 di-O-pivaloyl -6-[fluorescein-5-carboxamido]azidohexane 19 (0.30 g, 0.4 mmol) was dissolved in 1,4-dioxane (30 mL) and concentrated aqueous ammonia (35%, 60 mL) was added. After 3.5 h the reaction mixture was concentrated, co-evaporated with methanol (30 mL × 3) and purified by flash column chromatography, eluting with methanol in dichloromethane (0–30%) with 0.5% trifluoroacetic acid. The product was dissolved in methanol and precipitated by addition of water (15 mL). Drying overnight under vacuum gave compound 3 as an amorphous yellow powder (0.19 g, 93%). δΗ (400 MHz, DMSO-d6) 10.14 (2H, s, COOH), 8.79 (1H, t, J = 5.4 Hz, NH), 8.47 (1H,br. s, ArH), 8.25 (1H, d, J = 8.1 Hz, ArH), 7.37 (1H, d, J = 8.1 Hz, ArH), 7.00 (1H, br. s, OH), 6.70 (1H, s, ArH), 6.69 (1H, s, ArH), 6.61–6.52 (4H, m, ArH), 3.39–3.27 (4H, m, CH2NH and CH2N3), 1.62–1.49 (4H, m, CH2CH2NH and CH2CH2N3), 1.40–1.31 (4H, m, CH2CH2(CH2)2NH); δC (100.6 MHz, DMSO-d6) 168.1 (C), 164.4 (C), 159.5 (2C), 154.5 (C), 151.8 (2C), 136.3 (CH), 134.6 (CH), 129.0 (2CH), 126.4 (C), 124.1 (C), 123.1 (CH), 112.6 (2CH), 109.0 (2C), 102.2 (2CH), 83.2 (C), 50.5 (CH2), 37.8 (CH2), 28.7 (CH2), 28.1 (CH2), 25.9 (CH2), 25.8 (CH2); MS-ESI-: m/z 499.3 [M-H]-; HR-ES [M+H]+: m/z 501.1762; calcd. for C27H25N4O6 501.17741.

Synthesis, purification and analysis of PNA probes

Standard Fmoc PNA monomers and Fmoc Xal resin were purchased from Link Technologies Ltd; piperidine and DMF (peptide synthesis grade) were purchased from Rathburn; HATU and amino acids were bought from Novabiochem and CEM Ltd; DIPEA, 2,6-lutidine, acetic anhydride, trifluoroacetic acid (TFA) and triisopropylsilane (TIS) were bought from Sigma Aldrich. All the PNA oligomers were synthesized on an Expedite Nucleic Acid/PNA synthesizer on the 2 μmole scale. Cleavage and deprotection of the PNA was performed using TFA:water:TIS in a ratio of 95:2.5:2.5 (400 μL/2 μmole) for 20 min. Purification of PNA was performed by reversed-phase HPLC on a Gilson system using an Ascentis C18 (Supelco) column, 10 mm × 250 mm, pore size 10 micron. The following protocols were used: Run time 35 min, flow rate 3 mL per min, binary system, gradient: Time in min (% buffer B); 0 (5); 5 (5); 24 (30); 26 (95); 29 (95); 30 (5); 35 (5). Elution buffer A: 0.1% TFA in water and buffer B: 0.08%TFA in acetonitrile. Elution of PNAs was monitored by UV absorption at 295nm. PNAs were then freeze-dried and re-dissolved in water at working concentration. The molecular mass of the PNAs was verified by a Low Resolution MALDI-TOF MS on a Waters (micromass) TofSpec2E 337 nm laser operating in linear mode with external calibration of +/− 0.1%..

General procedure for solution phase click chemistry

A solution of CuSO4 in water (100 nmol / μL) was degassed by purging with argon gas and added to a degassed solution of sodium ascorbate (500 nmol / μL) in water. The mixture was degassed one more time before being added to a degassed solution of PNA in water. The 5-azidohexylfluorescein 3 in THF was added to the reaction mixture and the resultant mixture was degassed and left in the dark at room temperature for 2 h. THF 1 mL was added and the solution was centrifuged at 5000 rpm for 2 min. The supernatant was removed and the pellet was re-dissolved in 0.1% TFA in water. The product was purified by reverse phase HPLC. Note: The final concentration of PNA in the reaction was less than 0.2 mM, the ratio of water to THF was ~1:1) The molar ratio of PNA:CuSO4:sodium ascorbate:dye was 1:10:100:50.

General procedure for solid phase click chemistry

Use similar condition as for solution phase labeling, a solution of CuSO4 was added to a degassed solution of sodium ascorbate. The reaction was degassed one more time before being added to the PNA resin in DMF under argon. Then a solution of degassed dye in DMF was added to the reaction mixture under argon and the mixture was left at room temperature for 2 h. The resin was transfer to an ALL-FIT column (Link Technologies Ltd) and washed with DMF (4 × 1 mL), DCM (3 × 1mL) and diethyl ether (2 × 1mL). The resin was dried under a stream of argon gas for 10 min before cleavage using the same conditions as for the PNA probe synthesis.

PCR amplification and fluorescence melting analysis

Target amplification and melting curve analysis was performed using a Roche LightCycler® 1.5 instrument in single experiment. Reactions were performed in 10 μL reaction volumes, comprising 0.5 μM of reverse primer (RP), 0.05 μM of forward primer (FP), 0.5 mM dNTPs, 0.1 μM of the probe, 3.0 mM of MgCl2, 10 ng / µL BSA (Roche Diagnostics, Lewes, UK), 0.5 Units HotStarTaq (QIAGEN, Crawley, UK) and 4 ng of the DNA targets. The PCR thermal cycling conditions were 95°C for 15 min to activate the enzyme followed by 35 cycles of 94°C / 10 sec, 50°C / 15 sec and 72°C / 15 sec. Following amplification, reactions were heated to 95°C for 30 sec and cooled at 30°C for 1 min. Melting curve analysis was then performed by heating reactions from 30°C to 95°C at 0.1°C /sec. The fluorescence melting curve data were converted to the first derivatives giving the melting peaks (-d(F)/dT where F is fluorescence and T is temperature in°C). The total time of the amplification and melting curve analysis was on average 60 min and depended on the ambient temperature, i.e., ability of LightCycler® to cool to 30°C.

Oligonucleotide synthesis and purification

Oligonucleotides, including HyBeacons, were synthesized by standard methods.20

Acknowledgments

The authors wish to thank Julie M. Herniman for helping with MS analyses.

Glossary

Abbreviations:

Boc
tert-butyloxycarbonyl
tBu
tert-butyl
CuAAC
copper (I) catalysed alkyne azide cycloaddition
DCM
dichloromethane
DIPEA
diisopropylethylamine
DMAP
4-diamethylaminopyridine
DMF
N,N-dimethylformamide
EDC
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
EDTA
ethylenediaminetetraacetic acid
FAM
fluorescein
HATU
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate
HBTU
O-benzotriazole-N,N,N’,N’-tetramethyluronium hexafluorophosphate
HOBt
1-hydroxybenztriazole
hr
hour
NHS
N-hydroxysuccinimidyl
PCR
polymerase chain reaction
Piv
pivaloyl
PNA
peptide nucleic acid
PyBOP
benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate
RP HPLC
reversed-phase HPLC
rt
room temperature
SNP
single nucleotide polymorphism
TFA
trifluoroacetic acid
THF
tetrahydrofuran
TIS
triisopropylsilane
TLC
thin layer chromatography
Tm
melting temperature
Tosyl
p-toluenesulfonyl
UV
ultraviolet

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