Clearly the methodology developed here and demonstrated for
Clearly the methodology developed here and demonstrated for the detection of HER2 mRNA biomarker, is a universal platform applicable for the detection of any genetic material.
It is noteworthy that the dUHBI monomer preserves the natural H-bonding pattern of dU and can be used as a monomer in a solid-phase oligonucleotide synthesis, without additional need for post-synthetic modification of ON probes. This is the first study that shows the utility of a derivative of the chromophore of the green fluorescent protein (HBI) as an intercalator for the detection of
Reagents and solvents were purchased from commercial sour-ces and were used without further purification. All moisture sen-sitive reactions were carried out in flame-dried reaction flasks with rubber septa, and the reagents were introduced with a syringe. All reactants in moisture sensitive reactions were dried overnight in a vacuum oven. Progress of reactions was monitored by TLC on pre-coated Merck silica gel plates (60F-254). Visualization was accom-plished by UV light. Medium pressure chromatography was carried out using automated flash purification system (Biotage SP1 sepa-ration system, Uppsala, Sweden). Compounds were characterized by nuclear magnetic resonance using Bruker AC-200, DPX-300 and DMX-600 spectrometers. 1H NMR spectra were measured at 200, 300 and 600 MHz. Phosphoramidite monomer was characterized by 31P NMR in C6D6 on Bruker UltraShieldTM-500, at 203 MHz. Chemical shifts are expressed in ppm, downfield from Me4Si (TMS), used as internal standard. Compounds were analyzed under ESI (electron spray ionization) conditions on a Q-TOF micro-instrument (Waters, UK). Unmodified oligonucleotides were purchased from Integrated DNA Technologies (Coralville, Iowa, USA). The oligonu-cleotide ssON1 was synthesized by the standard automated solid-phase method, with a slightly adjusted protocol concerning the modified monomer 11, on an H-8 standard DNA synthesizer (K&A, Germany). MALDI-TOF mass spectra of oligonucleotides were measured with mass spectrometer in a negative Necrostatin 1 mode with THAP matrix. Absorption spectra were measured on a UV- 2401PC UVevis recording spectrophotometer (Shimadzu, Kyoto, Japan). Emission spectra were measured using Cary Eclipse Fluorescence Spectrophotometer. Absorption and fluorescence spectra were recorded in PBS buffer containing NaC1 8.0 g, KC1 0.2 g, Na2 HPO4 1.15 g, KH2PO4 0.2 g, in water 100 mL (pH 7.4).
5-(6-p-Methyl-ethyl-hydroxybenzylideneimidazolinone-1-hexene)-50-ODMTr-20-deoxyuridine 10 (180 mg, 210 mmol) was coevaporated in dry pyridine (2 mL), dry toluene (2 mL), dry acetonitrile (2 mL) and dry DCM (2 mL) and then dissolved in dry DCM (3 mL). DCI (248 mg, 210 mmol) and 2-cyanoethyl N,N,N0,N'-tetraisopropylphosphorodiamidite (76 mg, 80 mL, 0.25 mol) were added. The reaction mixture was stirred for 45 min at room tem-perature under argon atmosphere. The solvent was then evapo-rated under reduced pressure and the resultant crude material was purified by column chromatography using DCM: MeOH (30:1 þ 1% pyridine). The solvent was again evaporated under reduced pres-sure and the residue was dissolved in DCM (2 mL). This solution was added dropwise to hexane (20 mL) at 20 C. After centrifugation at 10 C and decantation the pellet was dissolved in DCM (2 mL), transferred to a Schlenk flask and the solvent was evaporated under reduced pressure to give 11 in 74% yield (165 mg). 31P NMR (203 MHz, C6D6): d 162.20, 161.62 ppm.
4.3. Synthesis of dUHBI-labeled oligonucleotide probes
For automated DNA synthesis, CPG support (200 nmol, pore size 500 Å, K&A Germany) and commercially available standard phos-phoramidites (Eurogentec) were used. Phosphoramidite 11 was dissolved in dry acetonitrile (1.5 mL, 0.1 M). For oligonucleotide synthesis, standard protocols were used to couple commercially available phosphoramidites. The coupling time for the 20-dUHBI monomer was prolonged from 2 to 5 min. As oxidizer, t-BuOOH in toluene (1.1 M) was used as a mild alternative to conventionally applied iodine standard solution. Cleavage of dUHBI-labeled oligo-nucleotide from the solid support was achieved by employing usual deprotection conditions, with 3:1 (v/v) NH3 (25%, aq.) and ethanol at 55 C for 20 h. The solvent was evaporated under reduced pres-sure and the residue dissolved in dry DCM (100 mL). Subsequently, desalination was performed using ethanol precipitation. For this, oligonucleotides in dry DCM (100 mL) were mixed with 10 mL so-dium acetate solution (3 M, pH 5.2). After the addition of ethanol (99%, 300 mL, chilled to 25 C), cooling the mixture in the freezer ( 25 C, 2 h) led to initial precipitation. A pellet was formed through centrifugation ( 4 C, 60 min, 13000 rpm) and washed thoroughly with ethanol (95%, 25 C). The thus obtained pellet was dried under reduced pressure. This crude product was purified by RP-HPLC on a C-18 column, and eluted with a linear gradient of 5e40% acetonitrile in 0.1 M TEAA (pH 7) over 30 min at a flow rate of 3 mL/min. The oligomer was converted into the sodium salt using CM Sephadex C-25 equilibrated in NaCl and washed well with water. The identity of the ON was determined by MALDI-TOF mass spectrometry: calcd for C211H266N72O125P20S ([MþNa])þ 6319.11, found 6319.19.