Probing RNA-Protein Interactions UsingPyrene-labeled Oligodeoxynucleotides: Qbbb ReplicaseEfficiently Binds Small RNAs by RecognizingPyrimidine Residues

Regine Preuû, Johannes Dapprich and Nils G. Walter*

Max-Planck-Institute forBiophysical ChemistryDepartment of BiochemicalKinetics, Am FassbergD-37077, GoÈttingen, Germany

Binding of small RNAs by the RNA-dependent RNA polymerase ofcoliphage Qb was studied utilizing a ¯uorometric assay. A DNA oligonu-cleotide probe of sequence 50-d(TTTTTCC) was 50-end-labeled with pyr-ene. In this construct, the proximal thymine residues ef®ciently quenchthe ¯uorophore emission in solution. Upon stoichiometric binding of oneprobe per polymerase molecule, the pyrene steady-state ¯uorescenceincreases by two orders of magnitude, the ¯uorescence anisotropyincreases, and a long ¯uorescence lifetime component of 140 ns appears.With addition of replicable RNA, steady-state ¯uorescence decreases in aconcentration dependent manner and the long lifetime component is lost.This observation most likely re¯ects displacement of the pyrene-labeledprobe from the proposed nucleic acid binding site II of Qb replicase. Theeffect was utilized to access binding af®nities of different RNAs to thissite in a reverse titration assay format. In 10 mM sodium phosphate (pH7.0), 100 mM NaCl, at 16�C, equilibrium dissociation constants for differ-ent template midi- and minivariant RNAs were calculated to be in thenanomolar range. In general, the minus and plus strands, concomitantlysynthesized by Qb replicase during replication, exhibited discriminativeaf®nities, while their hybrid bound less ef®ciently than either of thesingle strands. Different non-replicable tRNAs also bound to the poly-merase with comparable dissociation constants. By titration with DNAhomo-oligonucleotides it was shown that the probed site on Qb replicasedoes not require a 20 hydroxyl group for binding nucleic acids, but recog-nizes pyrimidine residues. Its interaction with thymine is lost in an A �Tbase-pair, while that with cytosine is retained after Watson-Crick base-pairing. These ®ndings can explain the af®nities of RNA-Qb replicaseinteractions reported here and in earlier investigations. The sensitivity ofthe described ¯uorometric assay allows detection of RNA ampli®cationby Qb replicase in real-time.

# 1997 Academic Press Limited

Keywords: ¯uorescence lifetime; ¯uorometry; MDV; real-time detection;RNA-dependent RNA polymerase*Corresponding author

Introduction

Qb replicase is the RNA-dependent RNA poly-merase responsible for replication of the single-stranded RNA genome of coliphage Qb. As inother viral RNA polymerases (isolated, e.g., fromphages MS2 (Fedoroff, 1975), GA (Yonesaki &Haruna, 1981), or SP (Miyake et al., 1971)), onlyone subunit of the heterotetrameric enzyme isphage encoded (Kamen, 1970; Kondo et al., 1970).This subunit renders replication by the enzyme

Present addresses: J. Dapprich, SEQ, SarnoffCorporation, Princeton, NJ 08540; USA; N. G. Walterc/o Professor John M. Burke, Department ofMicrobiology and Molecular Genetics, University ofVermont, Burlington, Vermont 05405, USA.

Abbreviations used: (�), plus strand; ds, double-stranded; Kd, equilibrium dissociation constant;MDV, midivariant RNA; MNV, minivariant RNA.

J. Mol. Biol. (1997) 273, 600±613

0022±2836/97/430600±14 $25.00/0/mb971343 # 1997 Academic Press Limited

speci®c to its own genome in the context of thehost's cellular RNA. The other three componentsare host encoded nucleic acid binding proteins,namely the ribosomal protein S1 (Wahba et al.,1974) as well as the elongation factors EF Tu andEF Ts (Blumenthal et al., 1972).

The exact roles of the host proteins for replica-tion of viral RNA by Qb replicase are yet to bede®ned. In Escherichia coli, EF Tu binds the 30 endof aminoacylated tRNAs to transfer them to theribosome for protein biosynthesis (Chinali et al.,1974). Hydrolysis of a bound GTP provides theenergy necessary for this process to take place. Asa subunit in Qb replicase, EF Tu seems to benecessary for RNA replication initiation only(Carmichael et al., 1976). Since replication initiateswith joining two molecules of GTP complementaryto the 30 end of the template, it has been suggestedthat EF Tu has similar roles both in protein biosyn-thesis and RNA replication: binding and bringingtogether GTP and the 30 end of an RNA molecule(Biebricher, 1983, 1987a). However, EF Tu has tobe complexed with EF Ts for full functionality inboth processes (Blumenthal & Carmichael, 1979).

The role of the protein S1 subunit of Qb replicaseis also obscure. During RNA replication, the plusstrand of the viral genome is bound by the poly-merase and used as a template for synthesis of thecomplementary minus strand, and vice versa. Thestrong secondary structure of the single-strandsimpairs double-strand formation, so that eachreplicative cycle roughly doubles the previousamount of single-stranded templates. The result isan exponential enrichment of replicated genomicand antigenomic RNAs (Dobkin et al., 1979;Biebricher et al., 1983, 1984). Ribosomal protein S1is necessary only for the synthesis of the minusstrand of the Qb genome. Another host factor isalso necessary for this step (Spiegelman et al.,1968), that binds the 30 end of the plus strand toenable replication initiation (Barrera et al., 1993).Thus, protein S1 might mediate the interaction ofQb replicase with this additional host factor(Biebricher, 1987a). Additionally, S1 has beenshown to bind two sites on the plus strand essen-tial for replication, prompting the notion of S1providing template recognition prior to comp-lementary strand synthesis (Senear & Steitz, 1976;Meyer et al., 1981). In contrast, the minus strand ofQb-RNA can be copied in vitro using a polymerasepreparation lacking both protein S1 and the hostfactor (Kamen et al., 1972). This observationsuggests that minus strand recognition is accom-plished by the three other subunits of the Qb repli-case complex alone.

More recently, a model for the nucleic acid bind-ing properties of the different subunits has beenproposed, based on in vitro selection of RNAligands and their UV-crosslinking to Qb replicase(Brown & Gold, 1995a, 1996). According to thismodel, the S1 protein binds a class I of RNAligands, including the plus strand of Qb-RNA, thatexhibits a high fraction of unpaired A and C bases.

A second binding site is located on EF Tu andinteracts with class II ligands with polypyrimidinestretches. This combination of two independentbinding sites would have the bene®t of facilitatingreplication by Qb replicase, since the polymerasecould stay in contact with both the template andthe product strand for a fast reinitiation of syn-thesis (Brown & Gold, 1996).

Apart from viral RNA (4217 nucleotides long),Qb replicase can utilize a number of short-chained(30 to 250 nt) templates for replication. In Qbphage infected Escherichia coli cells, a variety ofRNA variants summed up as ``6 S RNA'' is gener-ated in conjunction with Qb-RNA (Banerjee et al.,1969). Spiegelman (1971) and co-workers selectedsome ef®ciently replicated variants in in vitro evol-ution experiments starting from Qb-RNA. Numer-ous midi-, mini-, and nanovariants were generatedin vitro without the addition of an exogeneous tem-plate (Kacian et al., 1972; Sumper & Luce, 1975;Mills et al., 1975; Schaffner et al., 1977; Biebricheret al., 1981, 1982; Biebricher, 1987b; Munishkin et al.,1988, 1991; Biebricher & Luce, 1993; Moody et al.,1994). While Qb-RNA and the midivariant RNAshare a common internal sequence motif that isrecognized by the replicase (Nishihara et al., 1983),most replicable RNA variants do not show largesequence similarities. Therefore, considerableefforts have been made to ®nd a general basis forspeci®c RNA recognition by Qb replicase. Imagingby electron microscopy (Vollenweider et al., 1976;Barrera et al., 1993), footprinting experiments withRNases (Schaffner et al., 1977; Meyer et al., 1981),gel-retardation and ®lter retention assays (Werner,1991), deletion analyses (Schuppli et al., 1994), andin vitro selection for RNAs being bound (Brown &Gold, 1995a) or replicated by the enzyme (Zamoraet al., 1995; Brown & Gold, 1995b) have been uti-lized to characterize the RNA-protein interactionsin this model system.

We have devised a ¯uorometric assay employ-ing a 50-pyrene labeled DNA probe to study inter-actions of Qb replicase with various nucleic acids.By considering the probe sequence and length ofthe attachment linker, we were able to observe ¯u-orescence quenching through ¯uorophore-nucleobase interactions in the free probe. Despite itsdeoxyribose backbone, the probe is readily boundby the RNA-dependent RNA polymerase, asobserved through a profound ¯uorescence increaseupon addition of Qb replicase. With addition ofreplicable RNA to the probe-enzyme complex, the¯uorescence decreases again. Apparently, the bind-ing site on the enzyme is the same for both DNAprobe and RNA, resulting in displacement of theprobe from its shielding environment in the com-plex. This effect was used to access equilibriumdissociation constants of different RNAs andDNAs to Qb replicase in a reverse titration assay.The resulting data on af®nities of nucleic acid-Qbreplicase interactions provide insights intosequence speci®c recognition by this RNA poly-merase.

Pyrene-labeled DNA Probes for RNA Binding to Q� Replicase 601

Results

A 50-pyrene labeled DNA probe is boundand becomes dequenched byQbbb replicase

Pyrene-labeled DNA or RNA probes pre-viously have been used for studying nucleic acidinteractions. For example, the formation ofhybrids between complementary RNAs or DNAscould be monitored by exploiting a change inquenching ef®ciency of the attached pyrene(Koenig et al., 1977; Yamana et al., 1992a,b;Mann et al., 1992; Bevilacqua et al., 1992; Kierzeket al., 1993; Turner et al., 1996; Dapprich et al.,1997). The ¯uorophore is speci®cally quenchedthrough (possibly proton-coupled) electron-trans-fer interactions with proximal pyrimidine resi-dues, that are altered upon base-pair formation(Kierzek et al., 1993; Manoharan et al., 1995;Dapprich et al., 1997).

We have utilized this effect to design a readilyavailable DNA probe in which an attached pyr-ene moiety is highly quenched (Figure 1(a)). Wecoupled a pyrene butyric acid moiety via an ami-nopropyl linker to the 50 end of the DNAsequence 50-d(TTTTTCC), using standard syn-thesis chemistry (see Materials and Methods). Thepyrimidine-rich sequence and the medium lengthlinker favor quenching of the ¯uorophore by elec-tron transfer from the excited pyrene to the adja-cent bases (J.D., N.G.W. & C. Seidel, unpublishedresults). We found the probe steady-state ¯uor-escence decreased by about 180-fold as comparedto uncoupled pyrene butyric acid in aqueous sol-ution (data not shown).

The chosen probe is complementary to aninternal loop of the minus strand of MNV-11RNA (MNV-11(ÿ)), a short and highly structuredvariant replicated by Qb replicase (Figure 5). Sur-prisingly, addition of this RNA to the probeunder various conditions did not result in achange of the observed ¯uorescence signal (datanot shown). Apparently, either the formation ofprobe-RNA complex does not interfere with pyr-ene quenching or the complex does not formbecause of occlusion of the probe's potentialbinding site through the tight secondary and ter-tiary structure of the target RNA. However,when Qb replicase was added to the probe, thesteady-state ¯uorescence of pyrene increased bytwo orders of magnitude, and its emission spec-trum displayed a more pronounced vibrationalpeak pattern (Table 1 and Figure 2(a)). Concur-rently, the probe showed increased ¯uorescenceanisotropy and an additional long lifetime com-ponent of approximately 140 ns (Table 1 andFigure 2(b)). The pyrene moiety obviouslybecomes strongly shielded from quenching inter-actions with the attached nucleo bases in the pre-sence of enzyme, resulting in the observeddequenching (Figure 1(b)). These effects were notobserved with free pyrene butyric acid.

Figure 1. Molecular basis of the ¯uorescence assays.(a) Design of the DNA probe. A hepta-pyrimidine oligo-deoxynucleotide was linked via a 50-aminopropyl linkerto pyrene butyric acid as the detector ¯uorophore.(b) Binding of probe and competing nucleic acids to Qbreplicase. Pyrene (excited at 340 nm) is stronglyquenched through interaction with the pyrimidinesequence of the probe, when free in solution. With theprobe reversibly bound to the heterotetrameric replicaseof phage Qb, pyrene becomes dequenched and ¯uor-esces with a spectral peak at 385 nm. If nucleic acidligands to Qb replicase are added, they partiallyexchange with the bound probe, resulting in a ¯uor-escence decrease.

602 Pyrene-labeled DNA Probes for RNA Binding to Q� Replicase

Probe and Qbbb replicase form a stablestoichiometric complex, from which the probecan be displaced by replicable RNA

When an excess of double-stranded MNV-11RNA (MNV-11(ds)) was added to the mixture ofprobe with Qb replicase (Figure 1(b)), the enhancedpyrene steady-state ¯uorescence decreased againand its long ¯uorescence lifetime component wassuppressed (Table 1 and Figure 2(b)). MNV-11(ds)is known to bind to a site on the polymerase essen-tial for RNA replication, thereby inhibiting syn-thesis of the single-strands in late stages of in vitroreplication (Biebricher et al., 1984; Biebricher1987a).

Figure 1(b) offers an explanation for theobserved reversal of dequenching of the probe-Qbreplicase complex by addition of minivariant RNA(see Discussion). The pyrimidine-rich DNA probeis assumed to bind reversibly to Qb replicase at asite also utilized for binding replicable RNA.Through complex formation, the pyrene ¯uoro-phore is shielded from quenching interactions withthe probe's pyrimidine residues, resulting in a ¯u-orescence increase. Upon addition of replicableRNA, the probe is displaced from its binding siteby competition with the RNA, and it becomesquenched again.

To analyze the probe-Qb replicase interaction ina more quantitative way, we performed twotitration assays. First, 0.25 mM Qb replicase weretitrated with a stock solution of pyrene-labeledprobe. Fluorescence intensity was monitored as afunction of probe concentration to extract the equi-librium dissociation constant of the probe-enzymecomplex (Figure 3). Second, a reverse titration ofprobe-Qb replicase complex with MNV-11(ÿ)(Figure 5) was performed. The resulting ¯uor-escence decrease was used in a Scatchard plot toinfer the stoichiometry of RNA molecules requiredfor displacing the probe from the complex(Figure 4).

Equation (2) (see Materials and Methods) wasused to ®t the data points from the forwardtitration of Qb replicase with probe in Figure 3 toyield an equilibrium dissociation constant for the

enzyme/probe complex of 11 (�2) nM (Table 2).This value was reproducible in several titrationsmonitored either at an emission wavelength of385 nm or 416 nm. Noteworthy, the ¯uorescenceincrease in Figure 3 starts to level off at approxi-mately stoichiometric concentrations of probe andenzyme (0.25 mM each). This observation suggestsa 1:1 ratio of probe binding to Qb replicase.

Analyzing the ¯uorescence data from reversetitration of probe/Qb replicase complex withMNV-11(ÿ) in a Scatchard plot as described inequation (11) (see Materials and Methods) yieldeda linear regression line with a y-intercept of 0.93(Figure 4). This ®gure corresponds to the inverse ofthe number of binding sites on Qb replicase fromwhich MNV-(11) is displacing a pyrene-labeledprobe. Finding this number to be 0.93 stronglyargues for the scheme in Figure 1(b), where oneprobe molecule becomes bound per Qb replicasemolecule and can be displaced reversibly by replic-able RNA.

Equilibrium dissociation constants of nucleicacid-Qbbb replicase complexes from reversetitration assays

Using the difference in ¯uorescence of free50-pyrene-labeled probe versus probe bound toQb replicase, we were able to perform reversetitration assays for a variety of RNAs and DNAsthat would displace probe from its complex withQb replicase (Figure 1(b)). The most important ofthe employed replicable RNAs are depicted inFigure 5. Equation (9) (see Materials and Methods)was used to analyze the ¯uorescence titrationcurves by non-linear least-squares regression, asexempli®ed in Figure 6. The resulting dissociationconstants are listed in Table 2.

The dissociation constants (Kd) vary from 1.8 nMto 1200 nM, indicating a wide range of af®nitiesbetween nucleic acids and Qb replicase. Alongsidereplicable RNA, a variety of non-template nucleicacids, such as tRNAs or DNAs, are bound withconsiderable af®nity. The DNA probe binds to theenzyme with an intermediate Kd of 11 nM. This®nding is a prerequisite for accurately measuring

Table 1. Fluorescence intensities, anisotropies, and lifetimes of 50-pyrene-labeled probe 50-d(TTTTTCC), before andafter addition of Qb replicase and dsMDV-1 RNA

Samplea Relative intensity at 385 [416] nm Anisotropyc Lifetimes (ns) (rel. amplitudes)d

Probe 1 [1] ÿ0.061 0.73 [0.68], 6.39 [0.32]Probe � Qb replicase 121 [155] ÿ0.044 5.03 [0.07], 139 [0.93]Probe � Qb replicase �MDV(ds) Ðb ÿ0.039 0.30 [0.48], 8.25 [0.09], 142 [0.43]

a All measurements were performed in standard buffer (10 mM sodium phosphate, pH 7.0, 100 mM NaCl) at 16�C as described inMaterials and Methods. Concentrations were 1.5 mM probe and 1.0 mM Qb replicase for the intensity measurements, 5 mM probe foranisotropy and lifetime measurement of the probe alone, and 0.25 mM probe, 0.25 mM Qb replicase, and 1.0 mM MDV(ds) for theother anisotropy and lifetime measurements, respectively.

b A more detailed description of ¯uorescence intensities upon reverse titration of probe/Qb replicase complex with MDV(ds) isgiven in Figure 6.

c Fluorescence anisotopies were calculated from intensity measurements with polarized light as described in Materials andMethods.

d Data sets were ®tted as described in Materials and Methods to yield exponential decay components. Their amplitudes werenormalized to add up to 1.

Pyrene-labeled DNA Probes for RNA Binding to Q� Replicase 603

dissociation constants of other nucleic acids in areverse titration or displacement assay format. Ifthe probe bound too weakly to Qb replicase, thereverse titration curves would not change withnucleic acids of different af®nities, and a calcula-ted Kd would not re¯ect a valid estimate for thedissociation constant. Alternatively, if the probebound too tightly, it could not be displaced ef®-ciently by other nucleic acids. Thus, the short DNAprobe chosen here is not only favorable for ana-

lyses of nucleic acid/Qb replicase interactions dueto its ¯uorescence quenching properties, but alsobecause of a well suited dissociation constant.

Replication of RNA by Qbbb replicase can bemonitored in real-time by fluorescencedecrease of a pyrene-labeled DNA probe

Recently, Qb replicase has been suggested forexponential ampli®cation of reporter RNAs indiagnostic clinical assays (Chu et al., 1986; Kramer& Lizardi, 1989; Burg et al., 1995, 1996; Tyagi et al.,1996; Stone et al., 1996). The observation that a sen-sitive, easily synthesizable 50-pyrene-labeled DNAprobe can be used to detect binding of replicableRNAs to Qb replicase prompts a novel type of ¯u-orescence detection for these assays. We sup-plemented a standard ampli®cation reaction ofMNV-11 by Qb replicase with 0.25 mM pyrene-labeled probe (see Materials and Methods). Uponaddition of the probe, ¯uorescence increased asobserved before, due to probe binding to the poly-merase. With exponential ampli®cation of MNV-11RNA, the probe is increasingly displaced due tocompetition for the binding site of Qb replicase.Consequently, the pyrene ¯uorescence decreasessigni®cantly by about 60% (Figure 7). The timetrace of ¯uorescence thus shows an inverted pro®leas compared to detection by a free ¯uorophoresuch as ethidium bromide or propidium iodide,

Figure 2. Fluorescence properties of the 50-pyrene-labeled DNA probe, before and after addition of Qbreplicase and replicable RNA. (a) Steady-state ¯uor-escence emission spectrum of probe in standard buffer(10 mM sodium phosphate, pH 7.0, 100 mM NaCl) at16�C. After addition of stoichiometric amounts of Qbreplicase (continuous line), the probe shows enhanced¯uorescence as well as a more pronounced peak patternin comparison to probe alone (broken and dash-dottedlines). (b) Time-resolved ¯uorescence emission of probein standard buffer, at 16�C. Without further additives,the probe exhibits a rapid ¯uorescence decay (triangles).Upon addition of an equal amount of Qb replicase, alonger lifetime component appears (circles). Its contri-bution decreases again with addition of an excess ofMDV(ds) RNA (crosses). Fluorescence lifetimes andtheir amplitudes were derived from the indicated decaycurves (continuous lines) as described in Materials andMethods and are detailed in Table 1.

Figure 3. Titration of 0.25 mM Qb replicase with probein 10 mM sodium phosphate (pH 7.0), 100 mM NaCl, at16�C. Pyrene ¯uorescence increase over its initial valueI0 was monitored at 385 nm as a function of addedprobe concentration. A linear regression line from theinitial increase (broken line) was used to correlate themeasured ¯uorescence with the corresponding concen-tration of enzyme/probe complex [E �P] (dash-dottedlines to scale the right y-axis; see Materials andMethods). The data points were then ®tted withequation (2), as described in Materials and Methods(continuous curve), to yield an equilibrium dissociationconstant for the probe/Qb replicase complex of Kd � 11(�2) nM.

604 Pyrene-labeled DNA Probes for RNA Binding to Q� Replicase

which exhibits ¯uorescence enhancement uponintercalation into ampli®ed RNA (Schober et al.,1995; Burg et al., 1995).

Discussion

Fluorophore-labeled nucleotides and nucleicacids recently have found increasing interest forstudying nucleic acid/protein interactions (e.g.Allen et al., 1989; Giedroc et al., 1991; Guest et al.,1991; Delahunty et al., 1994; Carver et al., 1994;Huang & Klingenberg, 1995; Jezewska &Bujalowski, 1996; Thrall et al., 1996). We havedevised an assay in which the ¯uorescence of a50-pyrene-labeled DNA probe is enhanced upon 1:1stoichiometric binding to the RNA-dependentRNA polymerase of coliphage Qb. This effectseems to stem from shielding of the ¯uorophorefrom quenching effects by the pyrimidine residuesof the probe nucleic acid sequence (Manoharanet al., 1995; Dapprich et al., 1997). If nucleic acidsare added that compete for the same binding siteon Qb replicase, the probe ¯uorescence decreasesagain.

Upon combining probe and Qb replicase, theanisotropy of pyrene ¯uorescence increases fromÿ0.061 to ÿ0.044 (Table 1). This change is consist-ent with a probe/enzyme complex forming, thatcan be expected to result in a slower rotational dif-fusion and higher anisotropy of the dye on theprobe (Lakowicz, 1983). Moreover, a fast (< 1 ns)lifetime component of the free probe is lost withaddition of enzyme, while a long (�140 ns) lifetime

component appears. Upon addition of a fourfoldexcess of MDV(ds) RNA, the relative amplitude ofthe short lifetime component increases again, whilethat of the long lifetime component decreases(Table 1). Higher concentrations of competingRNA enhance this reversal (data not shown),resulting in the ¯uorescence decrease upontitration presented here. A reversible exchange ofcompeting nucleic acids on a single site of Qbreplicase (Figure 1(b)) seems the most likelyinterpretation of these ®ndings.

The probe ¯uorescence signal quickly (< ®veminutes) stabilizes after addition of Qb replicase,and again after addition of competing unlabelednucleic acids. Apparently, equilibrium adjustmentbetween free and Qb replicase-bound nucleic acidsoccurs rapidly. This ®nding is in agreement withthe fast association and dissociation rates pre-viously observed for MDV and Qb genomic RNAsbinding to the polymerase (Werner, 1991). WithQb replicase binding a variety of different nucleicacids, including tRNA and DNA, this fast

Figure 4. Scatchard plot of the ¯uorescence decreaseupon titration of probe/Qb replicase complex withMNV-11(ÿ) RNA. Equation (2) (see Materials andMethods) was used to calculate the parameters charac-terizing the interaction of MNV-11(ÿ) with polymerase.The slope of the linear regression line (continuous line,represented by the given equation with a correlationcoef®cient of r � 0.999) yields a dissociation constant ofKd � 4.5 nM, while the y-intercept de®nes the number ofprobe binding sites to n � 1/0.93 � 1.

Table 2. Dissociation constants (Kd) of nucleic acid/Qbreplicase complexes, as derived from forward andreverse titrations of 50-pyrene-labeled probe/enzymecomplexa

Nucleic acid Kd (nM) b

Probe 50-d(TTTTTCC) (11 � 2)MDV(ÿ) (3.5 � 0.7)MDV(�) (10 � 2)MDV(ds) (23 � 5)MNV-11(ÿ) (4.3 � 0.8)MNV-11(�) (17 � 5)MNV-11(ds) (24 � 8)SN0706 (5.1 � 0.9)SN0709 (5.2 � 0.9)DN3 (6.6 � 1.2)Total tRNA (E. coli) (38 � 7)Total tRNA (yeast) (20 � 4)Total tRNA (wheat germ) (10 � 2)tRNAPhe (E. coli) (360 � 65)tRNAfMet (E. coli) (31 � 6)tRNAVal (E. coli) (14 � 3)Synthetic tRNAAsp (E. coli) (11 � 2)Synthetic tRNASer (E. coli) (10 � 2)dC20 (1.8 � 1.2)dT20 (2.3 � 1.0)dG20 (1000 � 250)dA20 (1200 � 300)dC20/dG20(ds) (2.9 � 0.8)dT20/dA20(ds) (480 � 150)

a All measurements were performed in standard buffer (10 mMsodium phosphate, pH 7.0, 100 mM NaCl) at 16 oC as describedin Materials and Methods. Concentrations for the forward titra-tion were 0.25 mM Qb replicase in buffer titrated with a 7 mMprobe stock solution. For the reverse titration, a 17.5 mM RNAoder DNA stock solution was added gradually to buffer con-taining 0.25 mM Qb replicase and 0.38 mM probe.

b Kd values were derived from non-linear ®tting of titrationcurves as described in Materials and Methods. The given devia-tions re¯ect errors from at least two separate titrations, eachanalyzed at two emission wavelengths (385 and 416 nm). Theyalso take into account the error from determination of Kd forthe probe/enzyme complex. To obtain valid Kds for double-stranded RNA or DNA, it was ensured that the excess of oneof the single strands was less than 5% by puri®cation of dou-ble-strands on non-denaturing gels.

Pyrene-labeled DNA Probes for RNA Binding to Q� Replicase 605

Figure 5. Replicable RNA variants utilized in this study. MDV(�) is a naturally occuring RNA (Mills et al., 1973). Itsstructure is adapted from Nishihara et al. (1983). Bold letters indicate nucleotides that are identical with Qb minusstrand RNA. MNV-11 is a species that evolved in in vitro evolution experiments. Its plus and minus strands areampli®ed concomitantly by Qb replicase and can be replicated inde®nitely without selection of other variants(Biebricher, 1987b). SN0706 and SN0709 were derived from transcribed precursors after limited incubation with Qbreplicase (Zamora et al., 1995). DN3 evolved in an early stage of apparently template-free replication with Qb repli-case (Biebricher & Luce, 1993). The indicated secondary structures for MNV-11, SN0706, SN0709, and DN3 are pre-dicted by folding algorithms and were veri®ed by structure probing techniques (R. P. & C. K. Biebricher,unpublished results). However, it has to be emphasized that they represent only one of typically several alternativestructures, and that other structures might be prevalent in RNA preparations.

606 Pyrene-labeled DNA Probes for RNA Binding to Q� Replicase

exchange of bound nucleic acids might help theenzyme to screen for Qb genomic RNA in the cel-lular environment of infected E. coli. Of the manyindividual nucleic acid molecules encountering aninteraction with the polymerase, only those exhibit-ing additional properties such as a 50 leader stemand an unpaired C-tract on the 30 terminus willeventually be replicated (Zamora et al., 1995;Brown & Gold, 1995b).

On the basis of the recently proposed model fornucleic acid binding and replication by Qb repli-case, it seems reasonable to assume that the probebinds to the proposed RNA binding site II, pre-sumably located on subunit EF Tu (Brown & Gold,1996). This site has been shown to bind pyrimidinetracts of in vitro selected RNA ligands and assist incomplementary strand initiation (Brown & Gold,1995a, 1996). Despite its lack of 20-hydroxyl groups,the hepta-pyrimidine sequence of the probe(50-d(TTTTTCC)) appears to be suf®cient for bind-ing to this site with a nanomolar dissociation con-

stant (Kd � 11 nM). All nucleic acids withpyrimidines employed in the reverse titrationassay were able to displace the probe from this siteon Qb replicase. This observation is consistent withthe fact that most replicable RNAs preferably inter-act with binding site II, except for the Qb genomicplus strand (Brown & Gold, 1996).

Since only the binding of one probe moleculeper Qb replicase was detected by an increase inpyrene ¯uorescence, and since this molecule mostprobably binds to RNA binding site II, the equili-brium dissociation constant measured for theprobe/enzyme complex also re¯ects binding to siteII only. If more probe molecules bind to Qb repli-case, e.g. to site I, these additional molecules donot change their ¯uorescence signal upon binding.The same line of argumentation holds for thereverse titration assays, where the displacement ofonly one probe molecule per Qb replicase wasdetected by ¯uorescence quenching (Figure 4).Consequently, the equilibrium dissociation con-

Figure 6. Reverse titration of probe/Qb replicase complex with (a) single and double-stranded MDV RNAs, (b) singleand double-stranded MNV-11 RNAs, (c) short replicable variants, and (d) homo-oligodeoxynucleotides. For (a) to (c),both the data points for the ¯uorescence decrease with increasing RNA content as well as the curve ®ts usingequation (9) (see Materials and Methods) are shown. In (d), titration data points are connected for better visibility.

Pyrene-labeled DNA Probes for RNA Binding to Q� Replicase 607

stants obtained from this competition assay arevalid only for site II binding. In contrast to other,less speci®c techniques for measuring Kds ofnucleic acid/Qb replicase interactions such as gel-retardation or ®lter binding assays (Werner, 1991;Brown & Gold, 1995a), direct monitoring of bind-ing using the ¯uorescence assay described hereyields information on speci®c interactions with asingle site of Qb replicase.

Nanomolar equilibrium dissociation constantsfor replicable RNAs, as derived from pyrenequenching experiments, generally are consistentwith values obtained through gel-retardation or ®l-ter binding assays (Werner, 1991; Brown & Gold,1995a). However, MDV(�) has been described ear-lier to bind to Qb replicase with higher af®nitythan MDV(ÿ) (Werner, 1991), while in our studythey behaved the opposite under fairly similar con-ditions (Table 2). This discrepancy might relate tothe fact that binding RNA to all accessible sites onQb replicase as during gel-shifts or ®lter retentioncan only yield an average binding constant for allavailable sites, such as the suggested sites I and II.As described above, the ¯uorescence assay can beexpected to be more speci®c for site II binding.MDV(�) has been shown to bind to Qb replicasethrough interactions with nucleotide sequencesnear the middle of the RNA, that are almost identi-cal to an internal region of Qb antigenomic minusstrand (Figure 5; Nishihara et al., 1983). ThoughQb(ÿ) RNA appears to be a site II binder (Brown& Gold, 1996), it cannot be excluded that MDV(�)interacts with other regions as well. Anotherreason for the observed discrepancy in MDV bind-ing could be that the preparations used here and inthe earlier study displayed alternative secondary

structures depending on their thermal history.Indeed, different secondary structures have beenproposed for MDV(�) RNA (Nishihara et al., 1983;Chu et al., 1986). The observation that base-pairformation has a large impact on binding af®nitiesof pyrimidines, especially thymine or uracil, couldthen explain differences in performance of inde-pendent RNA preparations.

It has been known for some time that a broadclass of replicable RNAs exhibit pyrimidine-richtracts (Schaffner et al., 1977; Biebricher, 1987b;Munishkin et al., 1988, 1991; Moody et al., 1994).These sequence elements have been proposed toenable binding to Qb replicase (Sumper & Luce,1975) and more recently have been identi®ed ascharacteristic for site II ligands (Brown & Gold,1995a). The replicable RNAs employed in ourstudy all contain one or more such C/U-richdomains (Figure 5). The abundancy of theseelements and their accessiblity for the enzyme inthe secondary and tertiary structure context ofindividual RNAs will determine their distinct Kds.Discriminative binding af®nities for plus andminus strands, as synthesized concomitantlyduring replication (Biebricher et al., 1983, 1984),might then help in separation of the template fromthe product strands onto binding sites I and II, orin asymmetric ampli®cation. Interestingly, all threesmall RNAs derived from early stages of selectionexperiments (namely SN0706, SN0709, DN3;Figure 5), are binding with comparably high af®-nities. Ef®cient binding to Qb replicase thereforeappears to be an integral feature at this stage,before better replicable variants with additionalqualities like MNV-11 evolve.

Total tRNA from E. coli binds to Qb replicasewith lower af®nity than tRNA from eukaryoticsources (Kd � 38 nM versus 10 to 20 nM, respect-ively; Table 2). Individual tRNAs that are abun-dant in E. coli, such as tRNAPhe, bind with evenhigher Kd (360 nM for tRNAPhe). In addition, post-transcriptional modi®cations might play a role indetermining binding af®nities, since the twotRNAs used as unmodi®ed synthetic transcriptsexhibited higher af®nities than the natural tRNAutilized in this study (Table 2). Freshly synthesizedQb RNA will be unmodi®ed. It seems thereforeplausible that discriminative Kds may be an adap-tation of phage Qb to E. coli preventing excessivebinding to host tRNAs, which do contain a pro-truding C-rich 30 terminus, one of the prerequisitesfor binding and replication. Strikingly, tRNAAsp

and tRNAVal show signi®cantly higher bindingaf®nities (Kd � 11 nM in the case of synthetictRNAAsp, Kd � 14 nM with tRNAVal), and haveboth been suggested to act as templates in evol-ution of replicable RNAs from apparently tem-plate-free reactions. It was assumed that they areintroduced into these reactions as tightly bindingcontaminations of Qb replicase preparations(Munishkin et al., 1988; Moody et al., 1994).

Double-stranded replicable RNAs such as MDVand MNV bind signi®cantly weaker than their

Figure 7. Real-time monitoring of RNA ampli®cation byQb replicase. A standard replication reaction of MNV-11RNA was supplemented with 0.25 mM 50-pyrene labeledDNA probe as described in Materials and Methods. Asubsequent increase in ampli®ed RNA concentrationresulted in a decrease in ¯uorescence upon competitionof probe with MNV-11 RNA for binding sites on Qbreplicase.

608 Pyrene-labeled DNA Probes for RNA Binding to Q� Replicase

respective single-strands. However, because of thequasi-species nature of replicated RNAs (Rohdeet al., 1995), these preparations most likely rep-resent a mixture of partly mismatched double-strands, still displaying bulged, unpaired bases. Itis important to note that a C �G base-pair maintainsthe binding af®nity of an unpaired C base, while aT �A base-pair loses that of a T alone. This ®ndingsuggests that Qb replicase might display a hydro-gen bond acceptor in the major groove of adouble-stranded nucleic acid to interact with theexocyclic 4-amino group of cytidine. This inter-action would not be possible with the 4-keto groupof thymine or uracil, as it requires a hydrogenbond donor instead. For single-stranded nucleicacid binding, additional residues on pyrimidinescould be utilized for making contact with Qb repli-case, such as the 2-keto groups of both C and T orthe 3-imino and 3-amino groups of C and T,respectively. A discrimination against purines thenmight stem from their entirely different stereoche-mical demands.

In conclusion, the presented method for investi-gating nucleic acid/Qb replicase complexesemploying 50-pyrene-labeled DNA probes yieldsvaluable insights into af®nity and speci®city of theimplied interactions. Moreover, it enables a noveldetection method for diagnostic clinical assaysbased on ampli®cation of reporter RNA by Qbreplicase. The advantage of the latter is its sensi-tivity for speci®c RNA/polymerase interactions asopposed to simple quanti®cation of RNA as withintercalating ¯uorophores. Finally, because of theirsimple design and high sensitivity for changes intheir molecular environment, 50-pyrene-labeled oli-godeoxynucleotides could be adapted as probes forstudying other template/polymerase and, moregenerally, nucleic acid/protein interactions.

Materials and Methods

Materials

Qb replicase was isolated from an Escherichia colistrain carrying the phage encoded b subunit on a plas-mid (obtained from Dr M. A. Billeter, ETH ZuÈ rich),essentially as described previously (Sumper & Luce,1975) with minor modi®cations (Bauer, 1990). ReplicableRNA variants such as MDV-1 (Mills et al., 1973; Kramer& Mills, 1978) or MNV-11 (Biebricher et al., 1981, 1982;Biebricher, 1987b) were ampli®ed from stock solutionsby replication in 50 mM Tris-HCl (pH 7.5), 10 mMMgCl2, 0.1 mM dithiothreitol, 10% (v/v) glycerol,0.5 mM each ATP, CTP, GTP, UTP, and 100 nM Qbreplicase at 37�C. Double and single strands of the RNAvariants were prepared from the replication reactions byacrylamide gel electrophoresis in the presence of 1 mMMgCl2 as described (Mills et al., 1978; Biebricher et al.,1982). SN0706, SN0709 (Zamora et al., 1995), and DN3(Biebricher & Luce, 1993) were in vitro transcribed fromplasmids using T7 RNA polymerase. The majority oftRNAs were purchased from Sigma. tRNASer was in vitrotrancribed with T7 RNA polymerase from a PCR pro-duct encoding the Escherichia coli gene with an additional50 leading sequence of GGCAGCATGTCA. tRNAAsp was

a generous gift from C. S. VoÈrtler and F. Eckstein andhad been likewise transcribed from a plasmid encodingthe gene from E. coli. 20mer homo-oligodeoxynucleotideswere synthesized on a Milligene Expedite Synthesizerusing standard phosphoramidite chemistry from GlenResearch and deprotected according to the manufac-turer's instructions. The DNA probe of sequence50-d(TTTTTCC) was accordingly synthesized on anApplied Biosystems 392 DNA/RNA Synthesizer with a50-aminopropyl linker attached. After deprotection andHPLC puri®cation, the probe was coupled with pyrenebutyric acid succinimidylester (Molecular Probes,Eugene, OR) according to the manufacturer's instruc-tions, and again puri®ed by reverse phase HPLC toremove uncoupled DNA from the preparation. The con-centration of the 50 pyrene-labeled probe was deter-mined using an extinction coef®cient of 29,000 at awavelength of 346 nm, as found for pyrene attached topyrimidine bases (Kierzek et al., 1993).

Steady-state fluorescence measurements

Steady-state ¯uorescence spectra, intensities, and timetraces were recorded on a Perkin-Elmer LS5B spectro-¯uorometer in a thermostated quartz microcuvette (crosssection 3 mm � 3 mm). All measurements were per-formed in 10 mM sodium phosphate (pH 7.0), 100 mMNaCl as the standard buffer, at a solution temperature of16(�0.5)�C. Pyrene was excited at 340 nm (band width5 nm), and ¯uorescence emission for titration assays wasmonitored at both 385 nm and 416 nm (band width10 nm). Photobleaching of the ¯uorophore under theseconditions was found to be negligible.

Forward titration of Qb replicase with a 7-mM stocksolution of pyrene-labeled probe was performed with0.25 mM polymerase in 70 ml standard buffer. After eachaddition of probe, the solution was mixed gently toavoid denaturation of Qb replicase. Then the solutionwas incubated in the cuvette for ®ve minutes to ensureequilibrium adjustment before reading the ¯uorescenceintensity. At this point, the probe ¯uorescence hadbecome constant. The equilibrium dissociation constantfor the complex formation between enzyme (E) and pyr-ene-labeled probe (P), Kd,P, is then de®ned as:

Kd;P � �E��P��E � P� ���E�0 ÿ �E � P����P�t ÿ �E � P��

�E � P� �1�

with [E], [P], and [E �P] being the concentrations of freeenzyme, free probe, and the formed enzyme/probe com-plex, respectively. These concentrations can be derivedfrom the initial enzyme concentration [E]0 and the addedtotal probe concentration [P]t, as indicated. Equation (1)directly translates into:

�E � P� � Kd;P � �E�0 � �P�t2

ÿ�������������������������������������������������������������

Kd;P � �E�0 � �P�t2

� �2

ÿ�E�0�P�ts

�2�To infer the enzyme/probe complex concentration [E �P]from the ¯uorescence measurements, we assumed thatduring the initial steps of titration (with enzyme still inexcess over probe) the probe is virtually quantitativelyconverted into the complex. Moreover, a ¯uorescencecontribution of probe not bound to enzyme wasassumed to be negligible. Thus, a linear regression linefor the initial titration steps will link the measured ¯uor-escence to the corresponding concentration of enzyme/probe complex [E �P] (Figure 3). The data points for the

Pyrene-labeled DNA Probes for RNA Binding to Q� Replicase 609

titration could then be ®tted with equation (2), varyingonly parameter Kd,P.

Reverse titrations of the Qb replicase-probe complexwith different RNAs or DNAs was performed asdescribed above in 70 ml standard buffer with 0.25 mMpolymerase preincubated for 15 minutes with 0.38 mMpyrene-labeled probe. In the forward titration, this probeconcentration was high enough to saturate the ¯uor-escence signal (Figure 3). Typically, the RNA or DNAstock solutions employed for titration had a concen-tration of 17.5 mM, so that addition of 1 ml stock solutionwould correspond to one equivalent of Qb replicase.After gentle mixing and equilibrium adjustment for ®veminutes, the ¯uorescence emission was measured. Atitration with the same volume of water devoid ofnucleic acids was performed in parallel as a measure forsignal changes induced just by the mixing process.

The ¯uorescence of the reference decreased slightlyjust through titration with water, most probably due todenaturation and adsorptive losses of Qb replicase. Its¯uorescence value, Iref, can be expected to correspond tothe total enzyme/probe complex concentration, [E �P]t,that is available for complex formation in any given stepof titration. Moreover, during reverse titration with RNA(or DNA), this total enzyme concentration will be distrib-uted between complexes with both probe (E �P) andRNA (E �RNA), so that:

�E �RNA� � �E � P�t ÿ �E � P� �3�In the forward titration of enzyme with probe, the ¯uor-escence increase with enzyme excess is linear andbecomes saturated with 0.38 mM probe, the concentrationutilized in the reverse titrations. The ®rst observationsuggests a linear relation between measured ¯uorescenceintensity I and the enzyme/probe complex concentration(presumably both in the reference and the RNA-titratedsample), while the second argues for full saturation ofthe enzyme with probe before reverse titration starts.These two assumptions enable us to form equation (3)into:

�E �RNA� � Iref

Iref;0� �E � P�0 ÿ

I

I0�E � P�0 � �Iref;n ÿ In��E�0

�4�with indices 0 referring to initial values (before the ®rsttitration step) and n indicating the normalization withthe respective initial ¯uorescence value. With the de®-nition of the dissociation constants for probe, Kd,P, andRNA, Kd,RNA:

�E� � Kd;P�E � P��P� ; Kd;RNA � �E� � �RNA�

�E �RNA� �5�

one obtains the equation:

Kd;RNA � Kd;P

�RNA�t�IrefÿI� ÿ �E�0�P�0

I ÿ �E�0�6�

where [RNA]t, [E]0, and [P]0 are the total added RNAconcentration and the initial enzyme and probe concen-trations, respectively. Equation (6) was used to calculatevalues for Kd,RNA from single data values.

If V is the initial sample volume in the cuvette, x thevolume of added RNA stock solution of concentration[RNA]st, nP and nE the amounts of probe and Qb repli-case in the sample, and the normalized ¯uorescencedecrease of the reference, Iref,n, is represented by a ®rstorder decay as in equation (7), then equation (6) can be

formed into equation (8):

Iref;n � c� eÿxk �7�

I2nÿ�

Kd;P�RNA�stnE�Kd;RNA ÿ Kd;P� x� c� eÿ

xk

� Kd;RNA � nP

nE�Kd;RNA ÿ Kd;P��� In

� Kd;RNA � nP

nE�Kd;RNA ÿ Kd;P� c� eÿxk � 0 �8�

This second-order polynome yields the solution:

In � p

2�

��������������������������������������������������������������������p

2

� �2

ÿ Kd;RNA � nP

nE�Kd;RNA ÿ Kd;P� c� eÿxk

s�9�

with

p � Kd;P�RNA�stnE�Kd;RNA ÿ Kd;P� x� c� eÿ

xk � Kd;RNA � nP

nE�Kd;RNA ÿ Kd;P�The positive sign is valid for:

Kd;RNA < Kd;P

the negative one for:

Kd;RNA > Kd;P

Equation (9) was used to ®t the ¯uorescence data fromreverse titrations with Kd,RNA as the only parameter var-ied.

For determination of the available probe binding siteson Qb replicase, we chose a Scatchard plot. With nequivalent and independent binding sites, the fraction nof bound RNA molecules per enzyme molecule is givento:

n � n�E �RNA��E �RNA� � �E � P� �

n�Iref;n ÿ In�Iref;n

�10�

Using the same de®nitions as above, equation (10) leadsto:

1

n� 1

n� Kd;RNA

n� �P�0 ÿ In�E�0

Kd;P��RNA�t ÿ �Iref;n ÿ In��E�0�11�

By plotting:

1

n� Iref;n

Iref;n ÿ In

against:

�P�0 ÿ In�E�0Kd;P��RNA�t ÿ �Iref;n ÿ In��E�0�

the slope of a linear regression line will yield the dis-sociation constant, while the y-intercept will give theinverse of the number of probe binding sites n.

All data sets were ®tted with the appropriate equationusing Marquardt-Levenberg least-squares non-linearregression of the program Origin (MicroCal).

Finally, to monitor RNA synthesis by Qb replicase, astandard ampli®cation reaction with 50 mM Tris-HCl(pH 7.5), 10 mM MgCl2, 0.1 mM dithiothreitol, 10% gly-cerol, 0.5 mM each ATP, CTP, GTP, UTP, and 100 nMQb replicase in a 60 ml volume was supplemented with0.25 mM pyrene-labeled probe and initiated with 1 pMMNV-11(�) (Figure 5). For ef®cient replication, a reac-

610 Pyrene-labeled DNA Probes for RNA Binding to Q� Replicase

tion temperature of 37�C was chosen. Fluorescence read-outs were automatically recorded every four seconds.

Lifetime measurements

The ¯uorescence lifetime of pyrene was measured in acommercial single-photon counting device (EdinburghInstruments). A nitrogen discharge lamp at 337.2 nmwith a ¯ash frequency of 50,000 sÿ1 was used for exci-tation. The time between excitation pulse and emittedsingle-photon signal was measured by time-amplitude-conversion from a capacitor's potential into a multiplechannel analyzer. The signal of a scattering body wasused for deconvolution. No signi®cant difference in the¯uorescence decay curves could be detected with andwithout polarizers, proving that decay rates were notsigni®cantly in¯uenced by anisotropic effects. Allmeasurements were performed in 60 ml standard bufferat 16 oC over at least 20 minutes to ensure statistical sig-ni®cance. The probe and the Qb replicase-probe complexwere analyzed at concentrations of 5 mM and 0.25 mM,respectively. 1 mM MDV(ds) was added to obtain a sig-nal from a reverse titration-type of experiment. Data setswere ®tted by least-squares non-linear regression toyield up to three exponential decay components togetherwith their relative amplitudes.

Fluorescence anisotropy measurements

Depolarization of ¯uorescence is predominantlycaused by rotational diffusion of the ¯uorophore andtherefore re¯ects its mobility. The higher the ¯uorophoremobility is, the more depolarized its emission will be(Lakowicz, 1983). To analyze anisotropies of solutions asa measure for ¯uorescence polarization, 10 mm Glan-Thompson polarizers were used on an SLM 8000 spec-tro¯uorometer. Fluorescence intensities were measuredwith the polarizers for excitation (at 340 nm) and emis-sion (at 385 nm) subsequently in all four possible combi-nations of vertical (v, 0o) or horizontal (h, 90o) alignment,Ivv, Ivh, Ihv, and Ihh. Anisotropy A could then be calcu-lated as described (Lakowicz, 1983) from

A � �Ivv ÿ g� Ivh�=�Ivv � 2g� Ivh�; with g � Ihv=Ihh:

Acknowledgments

This work has been supported in part by a grantfrom the Bundesministerium fuÈ r Bildung und For-schung. The authors are very grateful to F. Salinquefor skillful technical assistance; to Dr E. Birch-Hirschfeld(Institute for Molecular Biotechnology Jena, Germany),Dr G. Kotzorek, and F. Benseler (NAPS GoÈ ttingenGmbH, Germany) for synthesis of pyrene-labeled oligo-deoxynucleotides; to Dr H. Staerk and B. Frederichs forhelp with the lifetime measurements; to M. Menger forhelp with the anisotropy measurements; to Dr G. Strunkand S. VoÈ lker for a gift of Qb replicase; to Dr P. Schwillefor advice on calculations; to Dr C. Biebricher andD. Vitiello for helpful comments on the manuscript; andto Dr M. Eigen and Dr J. McCaskill for a stimulatingresearch environment.

References

Allen, D. J., Darke, P. L. & Benkovic, S. J. (1989). Fluor-escent oligonucleotides and deoxynucleotide tripho-sphates: preparation and their interaction with thelarge (Klenow) fragment of Escherichia coli DNApolymerase I. Biochemistry, 28, 4601±4607.

Banerjee, A. K., Rensing, U. & August, J. T. (1969).Replication of RNA viruses. X. Replication of anatural 6 S RNA by the Qb RNA polymerase. J. Mol.Biol. 45, 181±193.

Barrera, I., Schuppli, D., Sogo, J. M. & Weber, H. (1993).Different mechanisms of recognition of bacterio-phage Qb plus and minus strand RNAs by Qbreplicase. J. Mol. Biol. 232, 512±521.

Bauer, G. J. (1990). Biochemische Verwirklichung undAnalyse von kontrollierten Evolutionsexperimentenmit RNA-Quasispecies in vitro. Dissertation, Techni-cal University Braunschweig, Germany.

Bevilacqua, P. C., Kierzek, R., Johnson, K. A. & Turner,D. H. (1992). Dynamics of ribozyme binding of sub-strate revealed by ¯uorescence-detected stopped-¯ow methods. Science, 258, 1355±1358.

Biebricher, C. K. (1983). Darwinian selection of self-repli-cating RNA. In Evolutionary Biology (Hecht, M. K.,Wallace, B. & Prance, G. T., eds), vol. 16, pp. 1±52,Plenum Press, New York.

Biebricher, C. K. (1987a). Replikation und Evolution vonRNA in vitro. Habilitation, Technical UniversityBraunschweig, Germany.

Biebricher, C. K. (1987b). Replication and evolution ofshort-chained RNA species replicated by Qbreplicase. Cold Spring Harbor Symp. Quant. Biol. 52,299±306.

Biebricher, C. K. & Luce, R. (1993). Sequence analysis ofRNA species synthesized by Qb replicase withouttemplate. Biochemistry, 32, 4848±4854.

Biebricher, C. K., Eigen, M. & Luce, R. (1981). Productanalysis of RNA generated de novo by Qb replicase.J. Mol. Biol. 148, 369±390.

Biebricher, C. K., Diekmann, S. & Luce, R. (1982). Struc-tural analysis of self-replicating RNA synthesizedby Qb replicase. J. Mol. Biol. 154, 629±648.

Biebricher, C. K., Eigen, M. & Gardiner, W. C. (1983).Kinetics of RNA replication. Biochemistry, 22, 2544±2559.

Biebricher, C. K., Eigen, M. & Gardiner, W. C. (1984).Kinetics of RNA replication: plus-minus asymmetryand double-strand formation. Biochemistry, 23,3186±3194.

Blumenthal, T. & Carmichael, G. G. (1979). RNA replica-tion: function and structure of Qb replicase. Annu.Rev. Biochem. 48, 525±548.

Blumenthal, T., Landers, T. A. & Weber, K. (1972). Bac-teriophage Qb replicase contains the protein biosyn-thesis elongation factors EF Tu and EF Ts. Proc.Natl Acad. Sci. USA, 69, 1313±1317.

Brown, D. & Gold, L. (1995a). Template recognition byan RNA-dependent RNA polymerase: identi®cationand characterization of two RNA binding sites onQb replicase. Biochemistry, 34, 14765±14774.

Brown, D. & Gold, L. (1995b). Selection and characteriz-ation of RNAs replicated by Qb replicase. Biochemis-try, 34, 14775±14782.

Brown, D. & Gold, L. (1996). RNA replication by Qbreplicase: a working model. Proc. Natl Acad. Sci.USA, 93, 11558±11562.

Burg, J. L., Cahill, P. B., Kutter, M., Stefano, J. E. &Mahan, D. E. (1995). Real-time ¯uorescence detec-

Pyrene-labeled DNA Probes for RNA Binding to Q� Replicase 611

tion of RNA ampli®ed by Qb replicase. Anal. Bio-chem. 230, 263±272.

Burg, J. L., Juffras, A. M., Wu, Y., Blomquist, C. L. &Du, Y. (1996). Single molecule detection of RNAreporter probes by ampli®cation with Qb replicase.Mol. Cell. Probes, 10, 257±271.

Carmichael, G. G., Landers, T. A. & Weber, K. (1976).Immunochemical analysis of the functions of thesubunits of phage Qb ribonucleic acid replicase.J. Biol. Chem. 251, 2744±2748.

Carver, T. E., Hochstrasser, R. A. & Millar, D. P. (1994).Proofreading DNA: recognition of aberrant DNAtermini by the Klenow fragment of DNA polymer-ase I. Proc. Natl Acad. Sci. USA, 91, 10670±10674.

Chinali, G., Sprinzl, M., Parmeggiani, A. & Cramer, F.(1974). Participation in protein biosynthesis oftRNA bearing altered 30-terminal ribosyl residues.Biochemistry, 13, 3001±3010.

Chu, B. C. F., Kramer, F. R. & Orgel, L. E. (1986). Syn-thesis of an ampli®able reporter RNA for bioassays.Nucl. Acids Res. 14, 5591±5603.

Dapprich, J., Walter, N. G., Salingue, F. & Staerk, H.(1997). Base-dependent pyrene ¯uorescence used forin-solution detection of nucleic acids. J. Fluoresc. 7,87S±89S.

Delahunty, M. D., Wilson, S. H. & Karpel, R. L. (1994).Studies on primer binding of HIV-1 reverse tran-scriptase using a ¯uorescent probe. J. Mol. Biol. 236,469±479.

Dobkin, C., Mills, D. R., Kramer, F. R. & Spiegelman, S.(1979). RNA replication: required intermediates andthe dissociation of template, product, and Qbreplicase. Biochemistry, 18, 2038±2044.

Fedoroff, N. (1975). Replicase of the phage f2. In RNAPhages (Zinder, N. D., ed.), pp. 235±258, ColdSpring Harbor Laboratory Press, Cold SpringHarbor, NY.

Giedroc, D. P., Khan, R. & Barnhart, K. (1991). Site-speci®c 1,N6-ethenoadenylated single-stranded oli-gonucleotides as structural probes for the T4 gene32 protein-22DNA complex. Biochemistry, 30, 8230±8242.

Guest, C. R., Hochstrasser, R. A., Dupuy, C. G., Allen,D. J., Benkovic, S. J. & Millar, D. P. (1991). Inter-action of DNA with the Klenow fragment of DNApolymerase I studied by time-resolved ¯uorescencespectroscopy. Biochemistry, 30, 8757±8770.

Huang, S. G. & Klingenberg, M. (1995). Fluorescentnucleotide derivatives as speci®c probes for theuncoupling protein: thermodynamics and kineticsof binding and the control of pH. Biochemistry, 34,349±360.

Jezewska, M. J. & Bujalowski, W. (1996). A generalmethod of analysis of ligand binding to competingmacromolecules using the spectroscopic signal orig-inating from a reference macromolecule. Appli-cation to Escherichia coli replicative helicase DnaBprotein-nucleic acid interactions. Biochemistry, 35,2117±2128.

Kacian, D. L., Mills, D. R. & Spiegelman, S. (1972). Areplicating RNA molecule suitable for detailed anal-ysis of extracellular evolution and replication. Proc.Natl Acad. Sci. USA, 69, 3038±3042.

Kamen, R. (1970). Characterization of the subunits of Qbreplicase. Nature, 228, 527±533.

Kamen, R., Kondo, M., RoÈmer, W. & Weissmann, C.(1972). Reconstitution of Qb replicase lacking sub-unit b with protein-synthesis interference factor i.Eur. J. Biochem. 31, 44±51.

Kierzek, R., Li, Y., Turner, D. H. & Bevilacqua, P. C.(1993). 50-Amino pyrene provides a sensitive, non-perturbing ¯uorescent probe of RNA secondaryand tertiary structure formation. J. Am. Chem. Soc.115, 4985±4992.

Koenig, P., Reines, S. A. & Cantor, C. R. (1977). Pyrenederivatives as ¯uorescent probes of conformationnear the 30 termini of polyribonucleotides. Biopoly-mers, 16, 2231±2242.

Kondo, M., Gallerani, R. & Weissmann, C. (1970). Sub-units structure of Qb replicase. Nature, 228, 525±527.

Kramer, F. R. & Lizardi, P. M. (1989). Replicatable RNAreporters. Nature, 339, 401±402.

Kramer, F. R. & Mills, D. R. (1978). RNA sequencingwith radioactive chain-terminating ribonucleotides.Proc. Natl Acad. Sci., USA, 75, 5334±5338.

Lakowicz, J. R. (1983). Principles of Fluorescence Spec-troscopy, Plenum Press, New York.

Mann, J. S., Shibata, Y. & Meehan, T. (1992). Synthesisand properties of an oligodeoxynucleotide modi®edwith a pyrene derivative at the 50 phosphate. Bio-conjugate Chem. 3, 554±558.

Manoharan, M., Tivel, K. L., Zhao, M., Na®si, K. &Netzel, T. L. (1995). Base-sequence dependence ofemission lifetimes for DNA oligomers and duplexescovalently labeled with pyrene: relative electron-transfer quenching ef®ciencies of A, G, C, and Tnucleoside toward pyrene. J. Phys. Chem. 99, 17461±17472.

Meyer, F., Weber, H. & Weissmann, C. (1981). Inter-actions of Qb replicase with Qb RNA. J. Mol. Biol.153, 631±660.

Mills, D. R., Kramer, F. R. & Spiegelman, S. (1973).Complete nucleotide sequence of a replicating RNAmolecule. Science, 180, 916±927.

Mills, D. R., Kramer, F. R., Dobkin, C., Nishihara, T. &Spiegelman, S. (1975). Nucleotide sequence ofmicrovariant RNA: Another small replicatingmolecule. Proc. Natl Acad. Sci. USA, 72, 4252±4256.

Mills, D. R., Dobkin, C. & Kramer, F. R. (1978). Tem-plate-determined, variable rate of RNA chainelongation. Cell, 15, 541±550.

Miyake, T., Haruna, I., Shiba, T., Itoh, Y. H., Yamane,K. & Watanabe, I. (1971). Grouping of RNA phagesbased on the template speci®city of their RNAreplicases. Proc. Natl Acad. Sci. USA, 68, 2022±2024.

Moody, M. D., Burg, J. L., DiFrancesco, R., Lovern, D.,Stanick, W., Lin-Goerke, J., Mahdavi, K., Wu, Y. &Farrell, M. P. (1994). Evolution of host cell RNAinto ef®cient template RNA by Qb replicase: the ori-gin of RNA in untemplated reactions. Biochemistry,33, 13836±13847.

Munishkin, A. V., Voronin, L. A. & Chetverin, A. B.(1988). An in vivo recombinant RNA capable ofautocatalytic synthesis by Qb replicase. Nature, 333,473±475.

Munishkin, A. V., Voronin, L. A., Ugarov, V. I.,Bondareva, L. A., Chetverina, H. V. & Chetverin,A. B. (1991). Ef®cient templates for Qb replicase areformed by recombination from heterologoussequences. J. Mol. Biol. 221, 463±472.

Nishihara, T., Mills, D. R. & Kramer, F. R. (1983). Local-ization of the Qb replicase recognition site in MDV-1 RNA. J. Biochem. 93, 669±674.

Rohde, N., Daum, H. & Biebricher, C. K. (1995). Themutant distribution of an RNA species replicatedby Qb replicase. J. Mol. Biol. 249, 754±762.

612 Pyrene-labeled DNA Probes for RNA Binding to Q� Replicase

Schaffner, W., RuÈ egg, K. J. & Weissmann, C. (1977).Nanovariant RNAs: nucleotide sequence and inter-action with bacteriophage Qb replicase. J. Mol. Biol.117, 877±907.

Schober, A., Walter, N. G., Tangen, U., Strunk, G.,Ederhof, T., Dapprich, J. & Eigen, M. (1995). Multi-channel PCR and serial transfer machine as a futuretool in evolutionary biotechnology. Biotechniques, 18,652±658.

Schuppli, D., Barrera, I. & Weber, H. (1994). Identi®-cation of recognition elements on bacteriophage Qbminus strand RNA that are essential for templateactivity with Qb replicase. J. Mol. Biol. 243, 811±815.

Senear, A. W. & Steitz, J. A. (1976). Site-speci®c inter-action of Qb host factor and ribosomal protein S1with Qb and R17 bacteriophage RNAs. J. Biol.Chem. 251, 1902±1912.

Spiegelman, S. (1971). An approach to the experimentalanalysis of precellular evolution. Quart. Rev. Bio-phys. 4, 213±253.

Spiegelman, S., Pace, N. R., Mills, D. R., Levisohn, R.,Eikham, T. S., Taylor, M. M., Peterson, R. L. &Bishop, D. H. (1968). The mechanism of RNAreplication. Cold Spring Harbor Symp. Quant. Biol. 33,101±124.

Stone, B. B., Cohen, S. P., Breton, G. L., Nietupski, R. M.,Pelletier, D. A., Fiandaca, M. J., Moe, J. G., Smith,J. H., Shah, J. S. & Weisburg, W. G. (1996). Detec-tion of rRNA from four respiratory pathogens usingan automated Qb replicase assay. Mol. Cell. Probes,10, 359±370.

Sumper, M. & Luce, R. (1975). Evidence for de novo pro-duction of self-replicating and environmentallyadapted RNA structures by bacteriophage Qbreplicase. Proc. Natl Acad. Sci. USA, 72, 162±166.

Thrall, S. H., Reinstein, J., WoÈhrl, B. M. & Goody, R. S.(1996). Evaluation of human immunode®ciencyvirus type I reverse transcriptase primer tRNAbinding by ¯uorescence spectroscopy: speci®cityand comparison to primer/template binding. Bio-chemistry, 35, 4609±4618.

Turner, D. H., Li, Y., Fountain, M., Profenno, L. &Bevilacqua, P. C. (1996). Dynamics of a group Iribozyme detected by spectroscopic methods. InNucleic Acids and Molecular Biology (Eckstein, F. &Lilley, D. M. J., eds), vol. 10, pp. 19±32, Springer-Verlag, Berlin.

Tyagi, S., Landegren, U., Tazi, M., Lizardi, P. M. &Kramer, F. R. (1996). Extremely sensitive, back-ground-free gene detection using binary probes andQb replicase. Proc. Natl Acad. Sci. USA, 93, 5395±5400.

Vollenweider, H. J., Koller, T., Weber, H. & Weissmann,C. (1976). Physical mapping of Qb replicase bindingsites on Qb RNA. J. Mol. Biol. 101, 367±377.

Wahba, A. J., Miller, M. J., Niveleau, A., Landers, T. A.,Carmichael, G. G., Weber, K., Hawley, D. A. &Slobin, L. I. (1974). Subunit I of Qb replicase and30 S ribosomal protein S1 of Escherichia coli. Evi-dence for the identity of the two proteins. J. Biol.Chem. 249, 3314±3316.

Werner, M. (1991). Kinetic and thermodynamic charac-terization of the interaction between Qb-replicaseand template RNA molecules. Biochemistry, 30,5832±5838.

Yamana, K., Gokota, T., Ozaki, H., Nakano, H., Sangen,O. & Shimidzu, T. (1992a). Enhanced ¯uorescencein the binding of oligonucleotides with a pyrenegroup in the sugar fragment to complementarypolynucleotides. Nucleosides Nucleotides, 11, 383±390.

Yamana, K., Ohashi, Y., Nunota, K., Aoki, M., Nakano,H. & Sangen, O. (1992b). Fluorescent-labeled oligo-nucleotides that exhibit a measurable signal in thepresence of complementary DNA. Nucl. Acids Res.Symp. Ser. 27, 135±136.

Yonesaki, T. & Haruna, I. (1981). In vitro replication ofbacteriophage GA RNA. Subunit structure and cata-lytic properties of GA replicase. J. Biochem. 89, 741±750.

Zamora, H., Luce, R. & Biebricher, C. K. (1995). Designof arti®cial short-chained RNA species that arereplicated by Qb replicase. Biochemistry, 34, 1261±1266.

Edited by I. Tinoco

(Received 27 May 1997; received in revised form 23 July 1997; accepted 8 August 1997)

Pyrene-labeled DNA Probes for RNA Binding to Q� Replicase 613