Synthesis of Aminoacylated tRNAThe pdCpA dinucleotide (tetrabutylammonium salt) and a sample of αOH-Phenylalanyl-dCA were obtained as a gift from Neurion Pharmaceuticals (Pasadena, CA). Subsequent preparation of αOH-Phenylalanyl-dCA was carried out according to the protocols in references 17-18. An example synthesis is described below.
Synthesis of αOH-Phenylalanine cyanomethyl ester (1)L-phenylactic acid (266 mg, 1.6 mmol) was dissolved in 3 mL DMF. To this was added chloroacetonitrile (3 mL, 47.4 mmol) and TEA (651 μL, 4.6 mmol). The reaction was allowed to proceed under nitrogen at room temperature overnight. The desired product was purified by flash chromatography (silica gel, 3:7 EtOAc:Hexanes). The final yield (amber oil) was 18.9 mg (68%
Synthesis of αOH-Phenylalanine-dCA (2)αOH-Phenylalanine cyanomethyl ester (11 mg, 54 μmol) was dissolved in 400 μL DMF and added to the tetrabutyl ammonium salt of pdCpA (9 μmol) in the presence of a catalytic amount of TBA-acetate. The reaction was allowed to proceed under nitrogen for 4 hr. at room temperature. The aminoacylated dinucleotide was purified by RP-HPLC using a gradient from 25 mM NH 4 OAc (pH=4.5) to CH 3 CN. Following lyophilization of the pooled fractions, the product was dissolved in 10 mM HOAc and lyophilized again. The final yield was determined by absorbance at 260 nm. and found to be 97 nmol (1%). The product was analyzed by ESI-
In vitro Transcription of THG73 tRNAThe plasmid harboring the THG73 gene was linearized with FokI and transcribed with T7 RNA polymerase. The transcription product was gel-purified by Urea-PAGE, dissolved in dH 2 O, and quantitated by absorbance at 260 nm.Ligation to THG73 tRNA 20 μg THG73 tRNA (8 μL in dH 2 O) in HEPES (22 μL, 10 mM, pH=7.5) was heated to 94 ºC for 3 min and allowed to cool slowly at room temperature. 8 μL αOH-Phenylalanine-dCA (3 mM in DMSO), 32 μL 2.5 X Reaction Buffer (25 μL 400 mM HEPES pH=7.5, 10 μL 100 mM DTT, 25 μL 200 mM MgCl 2 , 3.75 μL 10 mM ATP, 10 μL S1 5 mg/mL BSA, 26.25 μL dH 2 O), 7 μL water, and 4 μL T4 RNA Ligase (N.E.B.). After incubation at 37 ºC for 1 hr, the reaction was extracted once with an equal volume of phenol (saturated with 300 mM NaOAc, pH=5.0:CHCl 3 :isoamyl alcohol (25:24:1)) and ethanol precipitated. The pellet was washed with 70% EtOH, dried under vacuum, and dissolved in 1 mM NaOAc to a concentration of 2 μg/μL.
Construction of Fusion Templates (Phe(K), UAG(K), UAG(V))These dsDNA templates were created by PCR using overlapping primers: (Forward primer = Gen-FP: 5'-TAATACGACTCACTATAGGGACAATTACTATTTACAATTACA-3') and a unique reverse primer (Phe(K)- hr. The mRNA-DNA template was gel purified by Urea-PAGE, dissolved in water, and quantitated by absorbance at 260 nm.
Construction of MK(n) Library Templates (MK0, MK2, MK4, MK6, MK8, MK10)Antisense ssDNA templates were synthesized at the Keck Oligonucleotide Synthesis Facility (Yale). The sequences for the MK Library ssDNA templates are as follows: MK library dsDNA was amplified by PCR using the forward primer Gen-FP (5'-TAATACG...