End-labelling of RNA

In vivo or in vitro synthesised RNA generally contains a 5 -phosphate ) whereas RNA fragments generated by nuclease digestion usually contain a 3 -phosphate (Section 4.4). These phosphates must be removed prior to labelling of the respective ends. The same procedure may be used for dephosphorylations of either end. 

Calf Intestinal Phosphatase (RNase free 10 units/pl, Boehringer Mannheim) 

The dephosphorylation of in vitro transcribed RNA can be avoided if a diribonucleotides, such as CpG, ApG or UpG is included in the transcription mixture. The diribonucleotides will be incorporated at the 5 -end of the transcript (see Section 2.3.2). 

The end-labelled RNA can be purified in a denaturing polyacrylamide gel (n 1000) or an agarose gel (n 1000) or by gel filtration in Sephadex G-50. The phenol and chloroform extraction should be included after the gel filtration. If the RNA is used without purification a phenol and chloroform extraction should be included before the precipitation step. 

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V. Special applications of T4 PNK lacking 3 -phosphatase activity. This mutant enzyme is uniquely suitable for the preparation from 3 -NMP of 3, 5 -biphosphates which can be used for synthesis and labeling of DNA and RNA (38). The 5 -[ P]pCp is a reagent commonly used for the 3 -end labeling of RNA with the T4 RNA ligase (39). The 3 -labeled RNA can then be used for RNA sequencing and hybridization, as well as for the analysis of RNA structure by fingerprinting. 

Fig. 1. End labeling of eubacterial RNA with [a-MP]GTP in vitro. Deproteinized RNA preparations were incubated for 1 hr at 43 (lane 1) and 50° (lane 2) with (a-32P]GTP as described, then fractionated on a 5% polyacrylamide-8 M urea gel. Labeled fragments were visualized by autoradiography. Lane 1, Agrobacterium tumefaciens A136 lane 2, Azoarcus sp. BH72. Scale at right indicates position of Haelll restriction fragments of phage 4>X174 DNA in nucleotides.

Fig. 10. 5 End labeling of DNA and RNA. Labeling is most efficient with single- stranded molecules and at the 5 ends of double-stranded DNA with recessed 3 ends. Blunt-ended DNA and DNA with recessed 5 ends react more slowly. After removal of the S phosphate with alkaline phosphatase, the S OH group is rephosphorylated by the action of polynucleotide kinase and ATP. Alternatively, in the presence of excess ADP, the reverse reaction is encouraged, effectively leading to exchange between then 5 phosphate and the y-phosphate of ATP.

Alkaline phosphatase 1 Dephosphorylates 5 ends of RNA and DNA. Removal of S -P04 groups prior to kinase labeling to prevent self-ligation. 

To measure the folding equilibrium of the Tetrahymena L-21Sca ribozyme in various counterions, 32P-labeled ribozyme is prepared by T7 in vitro transcription with a-[32P]-ATP, according to established protocols (Zaug et al, 1988). Free label is removed with a size-exclusion spin column (e.g., TE-10, BD Sciences), and the RNA used without further purification (Emerick and Woodson, 1993). If desired, the RNA can be end-labeled with 32P and purified by denaturing PAGE. As for all biochemical experiments on RNA, care must be taken to use water, reagents, and plastic consumables that are free of RNase. 

Finally this sequencing method is not restricted to species for which abundant biological material is available 2- 3 /ig of unfractionated cellular RNA is usually sufficient to perform a reliable sequence determination from a given primer.13 In the protocol described below, which essentially applies to rRNA, radioactive cDNAs are generated by utilization of a 5 end-labeled primer rather than by incorporation of an a-labeled deoxynu-cleoside triphosphate (dNTP), in order to eliminate potential problems arising from self-primed reverse transcriptions. 

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