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RNA Sequencing with Radioactive Chain-Terminating Ribonucleotides

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RNA Sequencing with Radioactive Chain-Terminating Ribonucleotides Proc. Nati. Acad. Sci. USA Vol. 75, No. 11, pp. 5334-5338, November 1978 Biochemistry RNA sequencing with radioactive chain-terminating ribonucleotides (phage Qft replicase/MDV-1 RNA/3'-deoxyribonucleoside 5'4a-3Pltriphosphates/persistence of secondary structures during electrophoresis) FRED RUSSELL KRAMER AND DONALD R. MILLS Institute of Cancer Research and Department of Human Genetics and Development, College of Physicians and Surgeons, Columbia University, New York, New York 10032 Communicated by S. Spiegelman, August 14, 1978 ABSTRACT A rapid method for determining nucleotide Maxam and Gilbert (6): The DNA molecules are usually en- sequences in RNA is described. It employs the 3'-deoxy ana- zymatically labeled with 32P at their 5' ends, and fragment sets logues of the ribonucleoside triphosphates as specific chain terminators during RNA synthesis. For example, the inclusion are generated by preferential chemical cleavage. of 3'-deoxyuridine 5'-triphosphate in an RNA synthesis reaction Until now, only one approach has been explored for the rapid in addition to the four usual ribonucleoside triphosphate pre- sequencing of RNA: Homogeneous RNA molecules are enzy- cursors results in the synthesis of a set of ifferent-length matically labeled with 32P at their 5' ends, and fragment sets product strands that terminate in a 3-deoxyuridine that has been are generated by endonucleolytic cleavage (7, 8). This approach incorporated in place of uridine. To sequence an RNA, four separate reactions are run, each employing a different 3'-deoxy to RNA sequencing is limited by the specificity of the available terminator. Parallel electrophoretic analysis of the resulting four ribonucleases. Although endonucleases exist that are specific sets of specifically terminated product chains leads to a direct for guanosines (ribonuclease T1) and adenosines (ribonuclease reading of the nucleotide sequence. We tested this method by U2), there are no known ribonucleases that are specific for ur- sequencing MDV-1 (-) RNA, a molecule that is synthesized in idines or cytidines. Furthermore, the ribonucleases employed vitro by phage QjB replicase. The sequence read from the re- are single-strand specific, and thus have difficulty in generating sulting gels agreed completely with the known sequence of MDV-1 (-) RNA. The bands in some regions of the sequencing fragments in regions that form secondary structures. gels were unusually close to one another, as has also been ob- This paper describes another technique for sequencing RNA served in other rapid sequencing procedures, making order as- molecules. Fragment sets are generated in vitro by the presence signment in these regions very difficult. Because the secondary of specific chain-terminating ribonucleotide analogues during structure of MDV-1 (-) RNA was known, it was shown that the product synthesis. In addition, the chain-terminating nucleo- compression of the bands is due to the persistence of secondary tides are 32P labeled, resulting in the labeling of the structures during electrophoresis. Thus, structured regions of fragments nucleic acids may intr uce difficulties for sequencing tech- at their 3' ends as they are terminated. This method was used niques that employ the currently available methods of gel to sequence MDV-1 (-) RNA (9), a molecule that is synthesized electrophoresis. in nitro by QB replicase (10). Because the complete nucleotide sequence of MDV-1 (-) RNA had already been determined Rapid nucleic acid sequencing procedures depend on the (11) by classical procedures (12), we were able to test the ac- generation of a set of radioactive fragments of the molecule to curacy of the new method. Moreover, we were able to examine be sequenced. These fragments have a common 5' end and the effect of the presence of secondary structures in the RNA specifically terminate in one of the four bases. Four sets of on the sequencing procedure. Many secondary structures have specifically terminated fragments are prepared and electro- been identified and located in MDV-1 (-) RNA with the aid phoretically separated, in parallel, on polyacrylamide slab gels, of reagents (e.g., sodium bisulfite) that react specifically with under conditions designed to resolve chains differing in length nucleotides that are in a single-stranded conformation (un- by only one nucleotide (1). Because mobility in the gel is a published data). Although secondary structures had no effect function of chain length, the nucleotide sequence can be read on the generation of fragments, the results indicate that the directly from an autoradiograph of the gel. formation of structures in the terminated product chains does This approach was used by Sanger and his colleagues (2-5) influence their electrophoretic mobility. in the sequencing of DNA. Sets of DNA fragments were gen- erated in vitro by using DNA-dependent DNA polymerase. A MATERIALS AND METHODS common 5' end was ensured by the use of specific oligonu- cleotide primers (2). Specific termination at 3' ends was ac- Nucleotides. All four 3'-deoxyribonucleoside 5'-[a-32P]tri- complished in four separate ways: extension of partially syn- phosphates were custom synthesized at specific activities of thesized reaction products in the absence of one of the four 25-170 Ci/mmol (1 Ci = 3.7 X 1010 becquerels) by the Inter- nucleotide precursors (3), exonucleolytic cleavage of the par- national Chemical and Nuclear Corp., Irvine, CA. [a-32P]GTP tially synthesized reaction products (3), the presence of was purchased from the same company. Cordycepin triphos- chain-terminating analogues of the deoxyribonucleotides phate (3'-dATP) was obtained from Miles Laboratories. Unla- during the reaction (4), and the presence of ribonucleotides beled ribonucleoside triphosphates were purchased from P-L during the reaction, followed by alkaline hydrolysis of the re- Biochemicals. action products (5). In all of these methods, the product strands QB Replicase. This RNA-dependent RNA polymerase was were labeled by [a-32P]deoxynucleotides that were present at isolated from Qfl bacteriophage-infected Escherichia coli Q13 some time during product synthesis. DNA that is not synthe- by the procedure of Eoyang and August (13), with the hy- sized in vitro can be sequenced by a procedure developed by droxylapatite step omitted. MDV-1 RNA. "Midivariant" RNA was originally isolated The publication costs of this article were defrayed in part by page from a Qf replicase reaction that was not provided with ex- charge payment. This article must therefore be hereby marked "ad- ogenous template RNA (9). It consists of two antiparallel, sin- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate gle-stranded complements. Its biological origin and cellular this fact. function are not yet known. However, once it was isolated, it 5334 Downloaded by guest on September 30, 2021 Biochemistry: Kramer and Mills Proc. Natl. Acad. Sci. USA 75 (1978) 5335 was shown to be an excellent template for Q# replicase. The synthesis of MDV-1 RNA and the separation of the comple- mentary (+) and (-) strands by electrophoresis in the presence o 12 MLoI rj of Mg2+ ions has been described previously (14). In order to E 12_ 20Ji employ a homogeneous template in these experiments, we used E a mutant RNA whose sequence (15) differs from the published ._ 0 sequence of MDV-1 RNA (11) at positions 87 and 88 in the (-) 108 strand. For simplicity, this mutant RNA is referred to as CL "MDV-1 RNA" throughout this report. reaction mix- Sequencing Reactions. Four separate 100-IAI ) 6 tures were prepared, each containing: 84 mM Tris-HCl (pH U) 200 M 7.4), 12 mM MgC12, 100 ,M ATP, 100,uM UTP, 100,uM GTP, ji 100,uM UTP, 500MM of one of the four 3'-deoxyribonucleoside z 4 - [a-32P]triphosphates, MDV-1 (+) RNA template at 43 Asg/ml, and Qfl replicase at 61 tig/ml. Each reaction was incubated at 370C for 8 min and then terminated by bringing the mixture o 2 2,000 jjM to 1 mg of sodium dodecyl sulfate per ml, 10 mM EDTA, and 400 mM NaCl. Unlabeled yeast RNA from Calbiochem (10 Mg) was then added to each as carrier, and each mixture was ex- 0 2 4 6 8 10 tracted with an equal volume of phenol/cresol solution (16). Time (minutes) Deproteinization results in the annealing of partially synthe- FIG. 1. Inhibitory effect of 3'-deoxyadenosine 5'-triphosphate sized product strands to their template strands (17). Product on the synthesis of MDV-1 RNA by Qfl replicase. The concentration strands shorter than 10 nucleotides do not remain hybridized of adenosine triphosphate in each reaction was 200 ,gM. [a-32P]GTP to their templates and were lost during isolation. The RNA was was present to label the product strands. Samples of each reaction previously were taken every 30 sec, and the RNA was freed of unincorporated isolated by gel filtration and alcohol precipitation as nucleotides by absorption onto filter paper, followed by washing with described (15). The RNA from each reaction was dissolved in 300 mM trichloroacetic acid. The reaction conditions and the details 50 Ail of a solution containing: 7 M urea, 5 mM Tris-borate (pH of sample preparation have been described previously (15). 8.3), 200 MM Na2EDTA, and bromophenol blue and xylene cyanol tracking dyes (Eastman Organic Chemicals), each at 500 minate in a 3'-deoxyadenosine that had been incorporated in ug/iml. These solutions were then heated to 1000C for 60 sec, place of adenosine. By keeping the effective concentration of to melt the partially synthesized product strands free of their 3'-dATP low, compared with the concentration of ATP, ter- templates, and they were then chilled to 0°C. One- to 10-,Ml mination would be an infrequent event, and would occur with samples of each product (the volume dependent on the specific approximately equal probability at all adenosines in the se- activity) were then analyzed, in parallel, by electrophoresis on quence.
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