Synthesis of adenosine derivatives as transcription initiators and preparation of 5′ fluorescein- and biotin-labeled RNA through one-step in vitro transcription

Effects of N6- or 8-modification of adenosine nucleotides on transcription initiation under the T7 [phis]2.5 promoter. RNA was prepared in the absence (lane 1) or presence of 4 mM of either N6-HDA-AMP (lane 3) or 8-HDA-AMP (lane 5). Transcription solutions contained 0.25 mM ATP and 1 mM each of GTP, CTP, and UTP. In addition, ~0.1 μM of [α-³²P]ATP was included to label RNA products for visualization and quantitation. Samples were run to single-nucleotide resolution on an 8% denaturing polyacrylamide gel. Normal transcription produces three RNA bands (lane 1). There are N, N+1, and N+2 bands of RNA. After transcription, half of each RNA preparation was reacted with FAM-SE (lanes 2,4,6, 100 mM FAM-SE in 0.5 M NaHCO₃, 30 min at room temperature). Amino- and fluorescein-RNA yields are indicated (lanes 1–6). Compared with normal pppRNA (lane 1), the amino-RNA (lane 3) and FAM-labeled RNA (lane 4) migrate with slower rates, shifting upwards by roughly 1 and 2.4 nt, respectively.