Expedite 8909

DNA oligonucleotides were synthesized on an Expedite 8909 synthesizer (Applied Biosystems, Foster City, CA, USA) with the use of standard phosphoramidite chemistry and deprotected with the use of aqueous ammonia. RP-HPLC was used for purification, while gel-filtration through a Sephadex G25 column was used for desalting of the samples. The pH was regulated by addition of HCl and LiOH solutions and subsequent addition of buffer solution. If not stated otherwise, the samples in the presence of K⁺ ions were prepared at the final concentrations of KCl and potassium-phosphate buffer (pH 6.5) at 100 mM and 20 mM, respectively. The annealing of the samples before spectroscopic and PAGE analysis included 5 min incubation at 90 °C immediately followed by cooling in an ice-water bath for 20 min.

DNAzymes with either stem loop or hammer head conformation were designed for the selected exons (Fig. 2B) and the oligonucleotides were ordered from IDT. The LNA modified DNAzyme was made in-house by ABI Expedite 8909 nucleic acid synthesis system in 1 µM scale, deprotected by NH₄OH at 55 °C overnight and purified by NAP column (GE Health care).

F3B and the control sequence, bearing a 5′ hexylamino function, were synthesized on a 1 μmol scale with an ABI Expedite 8909 synthesizer, using conventional β-cyanoethyl phosphoramidite chemistry (2’OMe-purine and 2’F-pyrimidine). Once purified (electrophoresis on denaturating gels: 20% (19:1 acrylamide/bis-acrylamide), 7M urea, Tris-Borate-EDTA buffer), oligonucleotides were conjugated to DOTA or Cy5, according to a previously described protocol for MAG₃ coupling [33 (link)]. Briefly, 20 nmol of oligonucleotide were suspended in 100 μL of binding buffer (sodium bicarbonate/sodium carbonate 0.25 M, pH 8.3, sodium chloride 1 M, sodium ethylenediaminetetraacetate 1 mM) and gently stirred at room temperature. DOTA-NHS (Chematech^®) or Cy5-NHS (Interchim^®) (3 mg, in 30 μL of DMF) was added in portions at room temperature over 3 h. After complete addition, the suspension was stirred for an additional hour, and the crude was directly purified by HPLC (Macherey-Nagel Nucleodur column, 0.1 M triethylammonium acetate, pH 7.0, (acetonitrile/0.1 M triethylammonium acetate, pH 7.0: 80/20) gradient) to afford the oligonucleotide conjugates in 50−90% yield. Conjugate characterization was performed through MALDI-ToF mass spectrometry (Reflex III, Bruker) or Electrospray ionization (LCT Premier, Waters).