Double-stranded (ds)RNA is important
for a variety of biological systems.
The discovery of the dsRNA-binding
motif (dsRBM), coupled with the occurrence
of this motif in a wide variety
of functionally diverse proteins, has led
to increased interest and study of -
dsRNA (6,14). For example, the dsRNA-
activated protein kinase (PKR), an
enzyme involved in the cellular antiviral
response, contains two tandem copies
of the dsRBM. In addition, the dsRNA
adenine deaminases (dsRADs) contain
three tandem copies of this motif (7).
Likewise, the study of the RNA-dependent
RNA polymerase (RdRP) activity
associated with RNA virus transcriptases
and replicases also requires the
use of dsRNA. In each of these systems, the length of the typical RNA used is in
the 10–80 bp range (1,9)

Arnold, J.J.

Cameron, C.E.

Ghosh, S.K.B.

Carolina Digital Repository

de Lille, UMR CNRS 8527, 1 rue du Prof.Calmette 59000 Lille, France. Internet:anne-charlotte.savary@pasteur-lille.frReceived 18 February 1999; accepted10 June 1999.Anne-Charlotte Savary,Bertrand Georges and ClaudeAuriaultInstitut Pasteur-Institutde Biologie de LilleLille, FranceSingle-NucleotideResolution of RNAStrands in the Presence oftheir RNA ComplementsBioTechniques 27:450-456 (September 1999)Double-stranded (ds)RNA is impor-tant for a variety of biological systems.The discovery of the dsRNA-bindingmotif (dsRBM), coupled with the oc-currence of this motif in a wide varietyof functionally diverse proteins, has ledto increased interest and study of -dsRNA (6,14). For example, the dsR-NA-activated protein kinase (PKR), anenzyme involved in the cellular antiviralresponse, contains two tandem copiesof the dsRBM. In addition, the dsRNAadenine deaminases (dsRADs) containthree tandem copies of this motif (7).Likewise, the study of the RNA-depen-dent RNA polymerase (RdRP) activityassociated with RNA virus transcrip-tases and replicases also requires theuse of dsRNA. In each of these systems,BenchmarksVol. 27, No. 3 (1999)Figure 1. Schematic for the mechanism of trap strand invasion. Start: dsRNA products of the 3Dpolprimer-extension assay. Step 1: An excess of unlabeled RNA, trap strand, which is identical to that of theend-labeled RNA under investigation, is added to the dsRNA products in the presence of formamide.Step 2: Samples are heated to 65°C for 2–5 min resulting in the denaturation of the dsRNA products. Step3: Samples are removed from the heat and loaded on a denaturing polyacrylamide gel. The trap strandprecludes re-annealing of the end-labeled RNA.the length of the typical RNA used is inthe 10–80 bp range (1,9).Denaturing polyacrylamide gel elec-trophoresis (PAGE) of dsRNA frag-ments longer than 10 bp is prone to anumber of problems, most of whichstem from the high thermal stability ofRNA-RNA duplexes. While typicalconditions for denaturing PAGE (8 Murea and gel temperatures of 50°–60°C)are sufficient to denature and providesingle-nucleotide resolution for dsDNAand RNA-DNA hybrids, these condi-tions are not sufficient for comparableresolution of dsRNA. Temperatureshigher than 60°C induce hydrolysis ofRNA, and many dsRNA fragmentsmelt well outside the working range fordenaturing PAGE.Indeed, mechanistic studies ofRdRPs have been limited by the afore-mentioned problems. For example, a15-nucleotide (nt) RNA primer strandmigrates as a single, discrete band inthe absence of its complement (Figure2B, lane 1). However, this same 15-ntRNA primer strand migrates as a smearin the presence of a complementary 21-nt RNA template strand (Figure 2B,lane 2). As a result, most studies report-ed to date for this class of nucleic acidpolymerase have completely avoidedgel analysis and relied primarily on fil-ter-binding methods to follow RdRPactivity (2,4,5,8,10,11,16). In those in-stances in which primer extension hasbeen followed by PAGE, glyoxal treat-ment of RNA has been required to re-solve extended products (12). As it isessential to achieve single-nucleotideresolution of extended primers to quan-tify accurately product formation andto address specifically issues related topolymerase mechanism (1), an alterna-tive approach is warranted.We have developed a simple, effec-tive method for analyzing RNA by de-naturing PAGE in the presence of itscomplement. This method permits sin-gle-nucleotide resolution of dsRNA un-der the same conditions used for analy-sis of dsDNA and RNA-DNA hybridsBenchmarksVol. 27, No. 3 (1999)Figure 2. Primer extension by poliovirus RdRP, 3Dpol. (A) 15/21-mer primer/template used in thisstudy. (B) Time course for poliovirus RdRP-catalyzed incorporation of AMP into 15/21-mer using MnCl2as the divalent cation. Reactions contained HEPES, pH 7.5 (50 mM), 2-mercaptoethanol (10 mM), MnCl2(5 mM), ZnCl2 (60 µM), 3Dpol (5 µM), an end-labeled [32P]-15/21-mer primer/template (1 µM) and ATP(500 µM). Reactions were initiated by addition of 3Dpol and incubated at 30°C after which the reactionswere quenched at the indicated times by addition of EDTA to a final concentration of 50 mM. One micro-liter of the quenched reaction mixtures was added to 9 µL of loading buffer: 90% formamide, 50 mM Tris-borate, 0.025% bromophenol blue, 0.025% xylene cyanol and either no unlabeled 15-mer RNA (- TrapStrand) or 1 µM unlabeled 15-mer RNA (+ Trap Strand). Samples were heated to 65°C for 2–5 min beforeloading 5 µL on a 20% polyacrylamide gel containing 1× TBE (89 mM Tris, 89 mM boric acid, 2 mMEDTA, pH 8.0) and 7 M urea. Electrophoresis was performed in 1× TBE at 75 W. Gels were visualized bya Model 445SI PhosphorImager (Molecular Dynamics, Sunnyvale, CA, USA). The lane indicated by [-]is the labeled 15-mer primer strand alone. A detailed description of the methods used can be found in Ref-erence 1. The RNA used in this study was synthesized by Dharmacon Research (Boulder, CO, USA).by denaturing PAGE. The method re-lies on the addition of an excess of un-labeled RNA, the sequence of which isidentical to that of the end-labeledRNA under investigation. We refer tothis unlabeled RNA as the “trapstrand.” The trap RNA is added duringpreparation of samples for elec-trophoresis, thus requiring very little ofthis RNA. The trap strand is added inexcess of the labeled RNA to displacethis RNA and preclude re-annealing.The method is illustrated in Figure 1.The utility of this method for theanalysis of RdRP-catalyzed nucleotideincorporation is illustrated in Figure 2.The 15/21-mer primer/template used inthis study is shown in Figure 2A, andthe RdRP used was from poliovirus.Reactions were performed as describedin the legend to Figure 2, and productswere analyzed by phosphor imaging af-ter electrophoresis on a denaturing 20%polyacrylamide gel. In the absence of atrap strand, smears were observed, andin no case was single-nucleotide resolu-tion of the extended primer obtained(Figure 2B, lanes 2–6). However, in thepresence of the trap strand, single-nu-cleotide resolution of the extendedprimer was observed (Figure 2B, lanes8–17). These reactions were performedusing Mn++ as the divalent cation co-factor. In other polymerase systems,this cofactor has been shown to pro-mote mis-incorporation and incorpora-tion of nucleotide analogs, such asdideoxynucleotides (15). As only thesingle, correct nucleotide was providedin the experiment shown in Figure 2B,it seems reasonable to conclude that theuse of Mn++ as the divalent cation co-factor for 3Dpol also promotes mis-in-corporation. Of course, this effect ofMn++ on 3Dpol-catalyzed nucleotideincorporation would not have been un-covered without single-nucleotide reso-lution of the extended products.Taken together, the data presented inFigure 2 show that the thermal stabilityof dsRNA causes incomplete denatura-tion of the duplex, which substantiallylimits the resolution of individualstrands of RNA under standard condi-tions used for denaturing PAGE. How-ever, in the presence of a trap strand,modest heating of the sample (65°C)and a reasonable concentration of for-mamide in the loading buffer (80%) aresufficient to achieve single-nucleotideresolution of RNA by denaturingPAGE. It is worth noting that single nu-cleotide resolution was also achievedby using a 20% polyacrylamide gelcontaining 40% formamide and 7 Murea. However, as the length of dsRNAand/or GC content are increased, weanticipate that the effectiveness of for-mamide-urea gels will be diminished.In contrast, the effectiveness of a trapstrand should be independent of thelength or sequence of dsRNA used.BenchmarksMoreover, formamide-urea gels are dif-ficult to prepare, require fixing beforeexposure and are toxic.Two technical problems have limit-ed quantitative, mechanistic analysis ofRdRPs: (i) the limited availability ofhigh-quality, synthetic RNA and (ii) thepoor resolution of dsRNA fragments bydenaturing PAGE. The recent develop-ment of novel synthetic routes for pro-duction of RNA oligonucleotides (13)and the application of the methodologydescribed herein, provide a solution tothese problems. In addition, this meth-odology can be applied to the analysisof dsRNA fragments used in studies ofdsRBM-containing proteins and to theanalysis of exceptionally stable, GC-rich dsDNA or RNA-DNA hybrids. Forexample, addition of a trap strand canbe used to dissociate labeled RNA fromits complement in hydroxyl radicalmapping experiments in which labeledRNA is lost due to incomplete denatu-ration of duplex and exclusion of thisRNA from the gel (3). In addition,stops or compressions in known re-gions of DNA sequencing gels couldpotentially be removed by addition of aDNA trap strand.It is plausible that a DNA trap strandmay substitute for an RNA trap strand.This might serve to reduce the cost ofthe trap strand. However, the concentra-tion of a DNA trap strand might need tobe substantially greater than the corre-sponding RNA trap strand, owing to thereduced thermal stability of a RNA-DNA duplex relative to a RNA-RNAduplex. Of course, another alternative tochemically synthesized RNA thatwould diminish the cost of the trapstrand is enzymatically synthesizedRNA. As long as the transcription reac-tion is deproteinized, crude RNA tran-scripts should work well in this particu-lar application. In conclusion, the use ofa trap strand is a cost-effective, rapidand safe approach to improve the quali-ty of data obtained from denaturingPAGE analysis of radiolabeled, dsRNA.REFERENCES1.Arnold, J.J. and C.E. Cameron. 1999. Po-liovirus RNA-dependent RNA polymerase(3Dpol) is sufficient for template switching invitro. J. Biol. Chem. 274:2706-2716.2.Behrens, S.-E., L. Tomei and R. DeFrancesco. 1996. Identification and propertiesof the RNA-dependent RNA polymerase ofhepatitis C virus. EMBO J. 15:12-22.3.Bevilacqua, P.C. and T.R. Cech. 1996. Mi-nor-groove recognition of double-strandedRNA by the double-stranded RNA-binding do-main from the RNA-activated protein kinasePKR. Biochemistry 35:9983-9994.4.Buck, K.W. 1996. Comparison of the replica-tion of positive-stranded RNA viruses of plantsand animals. Adv. Virus Res. 47:159-251.5.Ishihama, A. and P. Barbier. 1994. Molecularanatomy of viral RNA-directed RNA poly-merases. Arch. Virol. 134:235-258.6.Kharrat, A., M. Macias, T.J. Gibson, M.Nilges and A. Pastore. 1995. Structure of thedsRNA binding domain of E. coli RNase III.EMBO J. 14:3572-3584.7.Kim, U., Y. Wang, T. Sanford, Y. Zeng andK. Nishikura. 1994. Molecular cloning ofcDNA for double-stranded RNA adenosinedeaminase, a candidate enzyme for nuclearRNA editing. Proc. Natl. Acad. Sci. USA91:11457-11461.8.Lohmann, V., F. Körner, U. Herian and R.Bartenschlager. 1997. Biochemical propertiesof hepatitis C virus NS5B RNA-dependentRNA polymerase and identification of aminoacid sequence motifs essential for enzymaticactivity. J. Virol. 71:8416-8428.9.Manche, L., S.R. Green, C. Schmedt andM.B. Mathews. 1992. Interactions betweendouble-stranded RNA regulators and the pro-tein kinase DAI. Mol. Cell Biol. 12:5238-5248.10.Neufeld, K.L., O.C. Richards and E. Ehren-feld. 1991. Purification, characterization, andcomparison of poliovirus RNA polymerasefrom native and recombinant sources. J. Biol.Chem. 266:24212-24219.11.Plotch, S.J., O. Palant and Y. Gluzman.1989. Purification and properties of poliovirusRNA polymerase expressed in Escherichiacoli. J. Virol. 63:216-225.12.Richards, O.C. and E. Ehrenfeld. 1998. Ef-fects of poliovirus 3AB protein on 3D poly-merase-catalyzed reaction. J. Biol. Chem.273:12832-12840.13.Scaringe, S.A., F.E. Wincott and M.H.Caruthers. 1998. Novel RNA synthesismethod using 5′-O-Silyl-2′-O-orthoester pro-tecting groups. J. Am. Chem. Soc. 120:11820-11821.14.St. Johnston, D., H. Brown, J.G. Gall andM. Jantsch. 1992. A conserved double-strand-ed RNA-binding domain. Proc. Natl. Acad.Sci. USA 89:10979-10983.15.Tabor, S. and C.C. Richardson. 1989. Effectof manganese ions on incorporation ofdideoxynucleotides by bacteriophage T7 DNApolymerase and Escherichia coli DNA poly-merase I. Proc. Natl. Acad. Sci. USA 86:4076-4080.16.Vázquez, A.L., J.M. Martín, R. Casais, J.A.Boga and F. Parra. 1998. Expression of enzy-matically active rabbit hemorrhagic diseasevirus RNA-dependent RNA polymerase in Es-cherichia coli. J. Virol. 72:2999-3004.This work was supported, in part, by aHoward Temin Award (CA75118) from theNational Cancer Institute (to C.E.C.) andNational Institutes of Health Grant No.GM58709 (to P.C.B.). Address correspon-dence to Dr. Craig E. Cameron, 201 Alt-house Laboratory, Biochemistry & Molecu-lar Biology, Pennsylvania State University,University Park, PA, 16802, USA. Internet:cec9@psu.eduReceived 24 March 1999; accepted 4June 1999.Jamie J. Arnold, SaikatKumar B. Ghosh, Philip C.Bevilacqua and Craig E.CameronPennsylvania State UniversityUniversity Park, PA, USABenchmarksVol. 27, No. 3 (1999)

Single-nucleotide resolution of RNA strands in the presence of their RNA complements

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Single-nucleotide resolution of RNA strands in the presence of their RNA complements

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