Design of thiazole orange oligonucleotide probes for detection of DNA and RNA by fluorescence and duplex melting† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c9ob00885c

We report fluorogenic duplex-stabilising thiazole orange (TO) functionalised oligonucleotides for nucleic acid detection in which TO is attached to the nucleobase or sugar of thymidine.


General synthetic procedures
All reagents were purchased from Sigma-Aldrich, Acros Organics, Lonza, Invitrogen or Fisher Scientific and used without further purification, or dried as described below.
3 Å molecular sieves (beads, 4 -8 mesh, Sigma-Aldrich) were used to dry MeOH and dry CH 2 Cl 2 was collected from a Grubbs-type SPS. Thin layer chromatography (TLC) was performed using Merck TLC silica gel 60 F 254 plates (0.22 mm thickness, aluminium backed) and the compounds were visualized by irradiation at 254/365 nm and stained with panisaldehyde or potassium permanganate. 1 H NMR spectra were measured at 400 MHz on a Bruker DPX400 (AVIIIHD 400) spectrometer. 13 C NMR spectra were measured at 101 MHz on a Bruker DPX400 spectrometer. 1 H were internally referenced to the appropriate residual undeuterated solvent signal; ¹³C NMR spectra were referenced to the deuterated solvent. Assignment of the signals was aided by COSY (¹H -¹H), HSQC-DEPT, HSQC (¹H -¹³C) and HMBC (¹H -¹³C) experiments.
Low-resolution mass spectra (LRMS) were recorded using electrospray ionisation (ESI + or ESI -) on a Waters ZMD quadrupole mass spectrometer in HPLC grade methanol.
High-resolution mass spectra (HRMS) were recorded in HPLC grade methanol using electrospray ionisation (EI) on a Bruker APEX III FT-ICR mass spectrometer. (1)  To a stirred solution of 1 (0.93 g, 2.7 mmol, 1.0 eq) and 2 (1.08 g, 3.3 mmol, 1.2 eq) in a mixture of anhydrous MeOH (34 mL) and anhydrous CH 2 Cl 2 (34 mL) under an argon atmosphere at RT, was added anhydrous Et 3 N (0.94 mL, 6.75 mmol, 2.5 eq). The solution turned red instantly and was then stirred for 15 min, precipitated with Et 2 O (400 mL), filtered and washed with Et 2 O (2 x 20 mL). The crude red solid was dissolved in THF (26 mL) and H 2 O (24 mL). LiOH (207 mg, 8.6 mmol, 2.5 eq) was added and the reaction mixture was stirred for 4 h. To the stirring suspension was added HCl (37%, 1.1 mL in 15 mL H 2 O) to give a red solution, the aqueous layer was extracted with CH 2 Cl 2 :2-propanol (v:v = 4:1; 2  40 mL).

Synthesis of DNA oligonucleotides
Standard DNA phosphoramidites, solid supports and additional reagents including amino-C6 dT phosphoramidite (C6, Figure S1

Synthesis of RNA oligonucleotides
2′-TBDMS (Tert-Butyldimethylsilyl) protected RNA phosphoramidite monomers with tbutylphenoxyacetyl protection of the A, G and C nucleobases were used purchased from Sigma-Aldrich and used to assemble RNA oligonucleotides. Benzylthiotetrazole (BTT) was used as the coupling agent, t-butylphenoxyacetic anhydride as the capping agent and 0.1 M iodine as the oxidizing agent (Sigma-Aldrich). Coupling time of 10 min was used and coupling efficiencies of >97% were obtained. Cleavage of oligonucleotides from the solid support and protecting groups from the nucleobase and backbone were removed by exposure to concentrated aqueous ammonia : ethanol (v:v = 3:1) for 2 h at room temperature followed by heating in a sealed tube for 2 h at 55 °C.

Removal of 2′-TBDMS protection of RNA oligonucleotides
After cleavage from the solid support and removal of the protecting groups from the nucleobases and phosphodiesters in ammonia/ethanol as described above, oligonucleotides were concentrated to a small volume in vacuo, transferred to 15 mL plastic tubes and freeze dried (lyophilised). The residue was dissolved in DMSO (300 μL) and triethylamine trihydrofluoride (300 μL) was added after which the reaction mixtures were kept at 65 °C for 2.5 h. Sodium acetate (3 M, 50 μL) and butanol (3 mL) were added with vortexing and the samples were kept at -80 °C for 30 min then centrifuged at 13000 rpm at 4 °C for 10 min. The supernatant was decanted and the precipitate was washed twice with ethanol (0.75 mL) then dried under vacuum. The fully deprotected oligonucleotides were purified in similar matter as DNA equivalents.

Synthesis of 2′-OMe-RNA oligonucleotides
Phosphoramidites, solid supports and additional reagents were purchased from Link Technologies, the procedure was identical to the DNA oligonucleotides apart from extended coupling time, 10 min, for each step including modifications.

Steady state fluorescence measurements
Fluorescence studies were performed on a Perkin Elmer LS50B luminescence spectrometer  (Table S8- 12). Examples of fluorescence emission spectra are given in Figure S22.

Method 1
Quantum yield measurements were made using UV-Vis and fluorescence spectrometers as

Method 2
The absolute fluorescence quantum yields were measured using a SC-30 integrating sphere module (Edinburgh Instruments) and the re-absorption effect was corrected when possible.     (Table S1) in the target strand counting from the TO-modified base, e.g. position "0" is directly opposite the TO modification and "-2" is two nucleobases towards the 5′ end of the probe sequence. ΔT m wasn't referenced as previously to completely unmodified duplexes due to lack of detectable T m of some of mismatched duplexes, data not

Oligonucleotide based
Ref.