Synthesis of adenosine derivatives as transcription initiators and preparation of 5' fluorescein- and biotin-labeled RNA through one-step in vitro transcription - PubMed (original) (raw)

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

Faqing Huang et al. RNA. 2003 Dec.

Abstract

Expanding our previous finding of an adenosine-initiated transcription system, we now demonstrate that either the 5' site or the N6 site of adenosine nucleotides can be modified extensively without abolishing their ability to initiate transcription under the T7 phi2.5 promoter. Two series of amino derivatives of adenosine nucleotides were synthesized. Fluorescein and biotin groups were coupled to AMP derivatives through linkers of different sizes and hydrophobicities. Both fluorescein- and biotin-conjugated (at either the 5' or N6 site) adenosine nucleotides can act as efficient transcription initiators, producing fluorescein- and biotin-labeled RNA at the specific 5' end by a one-step transcription procedure, eliminating posttranscriptional modification. Furthermore, N6-modified adenosine derivative-initiated transcription synthesizes 5' end modified RNA with a free phosphate group, providing the possibility for further derivatization. The current finding makes easily available a variety of site-specifically functionalized RNA, which may be used in nucleic acid detection, RNA structural and functional investigation, and generation and isolation of novel functional RNA.

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Figures

FIGURE 1.

Effects of N6- or 8-modification of adenosine nucleotides on transcription initiation under the T7 φ2.5 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.

FIGURE 2.

Synthetic scheme for amino derivatives of adenosine. (A) Synthesis of 5′ amino derivatives of adenosine phosphoramidate by EDAC-assisted direct coupling of diamines with AMP. (B) Preparation of N6-amino derivatives of AMP from 6-chloropurine riboside. Chlorophosphorylation of 6-chloropurine riboside followed by hydrolysis yields 6-chloropurine riboside 5′-monophosphate. Displacement of the 6-chloride by diamines produces the desired products. The linkers vary in length (3–14 bonds) and hydrophobicity. Abbreviations for diamines and their adenosine derivatives are in parentheses.

FIGURE 3.

Synthesis of fluorescein and biotin derivatives of AMP by reaction of amino derivatives of AMP with FAM-SE and biotin-SE. (A) Preparation of adenosine 5′-fluorescein- and 5′-biotin-phosphoramidates, and (B) synthesis of N6-fluorescein and N6-biotin derivatives of AMP.

FIGURE 4.

Transcription initiation with 5′- and N6-amino derivatives of adenosine nucleotides under the T7 φ2.5 promoter (top) and their posttranscriptional derivatization with fluorescein (bottom). RNA samples were run to single-nucleotide resolution on an 8% denaturing PAGE and visualized by phosphorimaging. Top panel: Transcription in the presence (4 mM) of one of the 12 amino derivatives produces high yields of 5′ end amino-modified RNAs, with migration rates ranging from slightly below that of the N+1 band to that of the N+2 band of unmodified RNA, depending on the linker size. Bottom panel: Coupling of fluorescein with 5′ end amino-modified RNAs further slows down the RNA migration in the gel by approximately 1 nt (i.e., with migration rates varying from slightly below that of the N+2 band to that of the N+3 band of unmodified RNA). Both amino-RNA yields and fluorescein-labeled RNA yields are indicated under the corresponding gels. The yields of fluorescein-linked RNA agree well with the results of amino-RNA in the top panel.

FIGURE 5.

Fluorescein-linked adenosine nucleotide-initiated transcription. RNA products were resolved by 8% denaturing PAGE and quantitated by phosphorimaging. Transcription in the absence (lane 1) or presence (2 mM) of fluorescein derivatives (5-isomers) of adenosine nucleotides (lanes 2_–_13). Transcription initiation efficiency varies from 0% to 65% (indicated under the gel, depending on the linker size and hydrophobicity) under the transcription conditions.

FIGURE 6.

Biotin labeling of RNA through 5′- and N6-biotin-modified adenosine nucleotides under the T7 class II promoter. Transcription was performed with either 0 mM (lane 1) or 4 mM biotin derivatives (lanes 2_–_8). Different biotinylated RNA products were resolved by 8% denaturing PAGE. Biotinylated RNA products comigrated with the N+2 and N+3 bands of unmodified RNA (depending on the linker size).

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Synthesis of adenosine derivatives as transcription initiators and preparation of 5' fluorescein- and biotin-labeled RNA through one-step in vitro transcription - PubMed (original) (raw)