Proc. Nat. Acad. Sci. USA Vol. 71, No. 12, pp. 5017-5021, December 1974 Deoxysubstitution in RNA by RNA Polymerase In Vitro: A New Approach to Nucleotide Sequence Determinations (pancreatic DNase I/U1 ribonuclease/U2 ribonuclease/pancreatic ribonuclease A/ribosubstituted DNA) GARY V. PADDOCK, HOWARD C. HEINDELL AND WINSTON SALSER Biology Department and Molecular Biology Institute, University of California at Los Angeles, 405 Hilgard Ave., Los Angeles, Calif. 90024 Communicated by Charles Yanofsky, August 12, 1974 ABSTRACT Deoxynucleotides have been incorporated substituted RNA. We have observed that Mn++ ion will not into RNA synthesized in vitro by RNA polymerase with only cause DNA polymerase to synthesize ribosubstituted either double-stranded or single-stranded DNA as a to synthesize template. By use of this technique to block or promote DNA but will also cause RNA polymerase cleavage at a particular phosphodiester bond, a variety of deoxysubstituted RNA. Subsequently we have discovered a specific cleavages may be obtained with the available ribo- number of other papers dealing with the synthesis of deoxy- nucleases and deoxyribonuclease I. These methods should substituted RNA (12-14). These papers did not, however, greatly increase the ease and rapidity of nucleotide se- point out the applicability of the approach to nucleotide quence determinations. sequencing and did not include the controls we have found The introduction of radiographic approaches by Sanger and essential for demonstrating that complete deoxysubstitution his colleagues (1-4) greatly increased the power of nucleotide has occurred. sequencing techniques, but there remain certain obstacles In this preliminary paper we discuss the theory by which whose solution could result in impressive further increases in substitution of deoxynucleotides becomes an aid in nucleotide the rapidity with which large sequences may be determined. sequencing, the technical precautions necessary to eliminate Examples of such problems, some merely irritating and others traces of contaminating ribonucleotides that would otherwise major, range from difficulties in ordering long tracts of pyr- be preferentially incorporated, and the evidence that the imidines to difficulties in obtaining good yields and purities of system preserves fidelity adequate for nucleotide sequencing the partial digestion products needed to obtain the order of studies. T1 or pancreatic RNase digestion products. In the case of ordinary RNA sequencing techniques the MATERIALS AND METHODS most serious difficulties spring from the lack of base-specific cleavages at nucleotides other than G which are needed to U1 RNase, DNase free, was the kind of gift of C. A. Dekker obtain overlaps. An improvement in this situation can be (15, 16), U2 RNase and T1 RNase were purchased from Cal- achieved by using ribosubstitution DNA sequencing tech- biochem. Spleen phosphodiesterase, pancreatic RNase A, and niques (5-9) in which one ribonucleotide is substituted for electrophoretically purified DNase I were purchased from one of the four deoxynucleotides during in vitro synthesis Worthington Biochemical Corp. This DNase I contained with DNA polymerase I. The product can then be degraded, RNase activity that was deactivated by treatment with so- cleaving at every ribose linkage by using alkaline hydrolysis dium iodoacetate (17). Hemoglobin (Hb) mRNA was sup- or the appropriate ribonuclease digestion. Unfortunately the plied by A. Bank and hemoglobin complementary DNA oligonucleotides resulting from such ribosubstitution cleavages (cDNA) was prepared and supplied by D. Kacian (18-20). cannot themselves be further cleaved at a specific base (in Dipodomys ordii (kangaroo rat) HS-,3 satellite DNA was pre- theory this could be achieved by synthesizing DNA sub- pared and supplied by J. Mazrimas and F. Hatch (21). Bac- stituted with two ribo-bases, such as G and C, but in fact the teriophage M13 DNA that had been depurinated and hy- rate of DNA synthesis is reduced to intolerably low levels drolyzed to give fragments of about 10 S was the gift of Kirk when this is attempted). Consequently such oligonucleotides Fry. must be sequenced using digestions with micrococcal nu- RNA polymerase prepared by the method of Burgess (22) clease (7, 9) or DNase I (unpublished work of B. Wallace in was a gift from C. Climenson and D. Eisenberg. a-82P- this lab) or by partial digestions with spleen or venom phos- labeled-nucleoside triphosphates were purchased from New phodiesterases (10, 11). England Nuclear Corp. and were purified of unidentified Ideally, one would like not only to be able to cleave spe- RNA polymerase inhibitors on a charcoal column (9). Non- cifically at several individual bases (as can be done by making radioactive nucleoside triphosphates were purchased from several ribosubstituted DNAs, each with a different ribo- Calbiochem and Sigma Chem. Co. We have found that it is base), but also to be able to cleave the same labeled product at extremely important to purify deoxynucleotides of contaminat- several different specific bases in successive digestions. This ing ribonucleotides, and for this purpose the method of Wu could be accomplished if it were possible to synthesize deoxy- (23) has proved convenient and effective. This is followed by purification on a DEAE-cellulose column with elution by tri- Abbreviations: cDNA, DNA complementary to RNA; Hb, ethylammonium bicarbonate, pH 8.5, in a 0.01 M-0.5 M gradi- hemoglobin. ent and subsequent desalting by passage through a Bio-Gel P2 5017 Downloaded by guest on October 2, 2021 5018 Biochemistry: Paddock et al. Proc. Nat. Acad. Sci. USA 71 (197 ) pH 3.5 Cellulose 6 Acetate---- 5 4 SIB 9 _wo . _ - - No XVU p P 7s!'I. B~~~~~~~OU (A I. 4t 2 * j p GGG 00U,_i2GA E SC (^"_GCCCC 33 (AG)U pyc -(AG% CCU * *2*201; I GU _6GC 9 6 w er6Cs B B- ST 5G : - a A F. B- .. sASC ANU.ALECTAG >FFACr ' 66. t~~~AAG#hi S Au j I VC'U t. ,.e It. d .. n of ,,paw. 9L.. a 9. 1A 1B 3A 3B 3C FIG. 1. Autoradiographs of two-dimensional fingerprints of Hb RNA. [a-32P]UTP-labeled Hb RNA (Fig. 1A, far left) and [a-32P]- UTP-labeled dC-substituted Hb RNA (Fig. 1B, middle left) were digested with U1 RNase and bacterial alkaline phosphatase and the products were separated by electrophoresis in two dimensions by the techniques described in Sanger et al. (1). The oligonucleotides are labeled as in Poon et al. (24). B is the blue dye marker and P is the pink dye marker. The origin is denoted by X. FIG. 3. Aatoradiographs of two-dimensional fingerprints of Hb RNA. [a.32P]CTP-labeled Hb RNA (Fig. 3A, middle); [a-32P]CTP- labeled, dT-substituted Hb RNA (Fig. 3B, middle right); and [a-32P]dCTP-labeled Hb RNA (Fig. 3C, far right) were digested with pancreatic RNase A and the products were separated by electrophoresis in two dimensions by the techniques described in Sanger et al. (1). 2':3'-Cyclic phosphates are denoted by > Oligonucleotides in Fig. 3C with the mobilities of U-Up, U-Up, and U-U-Up may have arisen from incomplete digestion of poly(U) by pancreatic RNase A. The poly(U) was labeled by [a-32P]rUTP contaminant. column. The 32P-labeled deoxynucleotides obtained from New were present. The deoxysubstituted RNA was subjected to England Nuclear Corp. were used without further treatment. hydrolysis overnight at 370 with 0.2 N LiOH and the diges- Synthesis of 32P-labeled Hb RNA was carried out as in ref. tion products were separated by electrophoresis on DEAE 24. Synthesis of deoxysubstituted RNA was carried out in paper at pH 3.5 or with 7% formic acid buffer. Each of the 100 Mul of 0.1 M Tris * HCl, pH 7.5, 2.5 mM MnCl2 containing digests was found to contain ribo-mononucleotides plus approximately 10 ,ug of RNA polymerase, 1 ,ug of DNA, 20 larger fragments. For example, when deoxy-A-substituted nmol of each ribonucleoside triphosphate, and 100 nmol of RNA was synthesized using [a-32P]GTP, radioactivity was the selected deoxynucleoside triphosphate except in the case found not only in Cp*, Gp*, and Up* but also in dAp*Gp, of the nucleoside triphosphate carrying the radioactive label. dApdAp*Gp, as well as lesser amounts of label in dApCp* 32P-Labeled nucleoside triphosphate (1 mCi) with a specific and dApdApCp* and dApUp*. Higher homologues (with activity of approximately 100 Ci/mmol was used per syn- more dA residues) also appeared to be present but were not thesis. The reaction mixture was made 0.2 M in KCl when further characterized. These results were confirmed in sep- double-stranded HS-3 DNA was the template. Incubation arate experiments in which the radioactivity was introduced was for 45 min at 37°. The product was then desalted and on the deoxynucleoside triphosphate. purified of nucleoside triphosphate precursors by passage of deoxysubstitution synthesis through a Bio-Gel P-60 column. (See Note Added in Proof.) Fidelity Nucleotide sequencing techniques and ribonuclease diges- These results suggested that it would be possible to achieve tions were those described in Sanger et al. (1), Barrell (2), the desired specifications of cleavage by using the deoxysub- Fry et al. (7), and Whitcome et al. (9). Digestion with pan- stitution technique. For example, we hoped that RNA deoxy- creatic DNase I was with 10 of 1.25 mg/ml of enzyme in substituted at C residues could be cleaved specifically at U by 0.1 M Tris HCl, pH 7.0, 0.02 M MgCl2, 2 mM CaCl2 over- treatment with pancreatic RNase or specifically at dC by night at 370 with 0l/ug of carrier RNA added. treatment with DNase I. Regardless of the apparent facility with which deoxysubstituted RNAs can be specifically RESULTS cleaved, the technique would be useless unless the fidelity of Incorporation of deoxynucleotides synthesis under deoxysubstitution conditions is good so that In our initial experiments we measured the ability of Esche- the correct sequences are obtained.