Acy ctp

DNA was transcribed in 100 μl transcription buffer as aforementioned except that DTT was excluded and 1 mM SBED-GMP^{8 (link)} was added to the transcription buffer. After transcription at 37 °C for 1 hour, the samples were irradiated and subjected to primer extension as the previous studies^{1 (link),4 (link)}. DNA was purified by Wizard SV Gel and PCR Clean-Up System (Promega) followed by primer extension with Deep Vent (exo-) (NEB, M0259) and FAM-5’-TGACAGCGATGCGTAGAATCGCTAG. The G ladder was obtained by extension of non-transcribed DNA in the presence of Acy-CTP (NEB, N0460).

Transcription was carried out as aforementioned but with the normal UTP being substituted with 1 mM of 4-S-UTP (TriLink BioTechnologies, CA, USA). Hundred microliters of transcribed and RNase H-digested DNA was subjected to UV-crosslinking as described (13 (link),19 (link)). The DNA was then treated by RNase A and EDTA as aforementioned, recovered with phenol/chloroform extraction and purified by the TIANquick mini purification kit (Tiangen, Beijing). Primer extension was performed with 0.4-μM 5′-FAM-CCAGCCTGCGGCGAGTG primer, 4 U of Deep VentR DNA polymerase (exo-) (NEB, MA, USA) in a 50-μl volume containing 75-mM Tris-HCl, pH 8.8, 20-mM (NH₄)₂SO₄, 0.1% (v/v) Tween 20, 2-mM MgCl₂, 0.05-mM dNTP and 5% (v/v) DMSO. G and T ladders were synthesized in the same way using a non-transcribed and non-crosslinked DNA strand in the presence of Acy-CTP and Acy-ATP (NEB), respectively, in a molar ratio of 1/2 and 1/1 to dCTP and dATP, respectively.