Interaction between hammerhead ribozyme and RNA substrates measured by a surface plasmon resonance biosensor

J. Biochem. Biophys. Methods 44 (2000) 41–57 www.elsevier.com / locate / jbbm

Interaction between hammerhead ribozyme and RNA substrates measured by a surface plasmon resonance biosensor ˚ Bandholtz a , ¨ a,1 , Asa Tommy Nyholm a,1 , Michael Andang a d ¨ Persson , Graham Hotchkiss a , Catharina Maijgren , Bjorn c a a,b , ¨ * Thomas E. Fehniger , Sten Larsson , Lars Ahrlund-Richter b

a Department of Medical Nutrition, Karolinska Institutet, Huddinge S-141 57, Sweden Department of Bioscience at Novum, Karolinska Institutet, Huddinge S-141 57, Sweden c Division of Clinical Virology, Department of Immunology, Microbiology, Pathology and Infectious Disease, Sweden, S-754 50 Uppsala, Sweden d Biacore AB, Sweden, S-754 50 Uppsala, Sweden

Received 25 October 1999; received in revised form 9 December 1999; accepted 9 December 1999

Abstract Dynamic interactions between hammerhead ribozymes and RNA substrates were measured using the surface plasmon resonance (SPR) technology. Two in vitro transcribed substrates (non-cleavable and cleavable) were immobilised on streptavidin-coated dextran matrices and subsequently challenged with non-related yeast tRNA or two hammerhead ribozymes, both of which had previously been shown to exhibit functional binding and cleavage of complementary target RNAs. The target-binding domain of one of the ribozymes was fully complementary to a 16-ribonucleotide stretch on the immobilised substrates, while the other ribozyme had a nineribonucleotide complementarity. The two ribozymes could readily be differentiated with regard to affinity. Cleavage could be measured, using the ribozyme with full target complementarity to the cleavable substrate. In contrast, the ribozyme with lower affinity lacked cleavage activity. We suggest that SPR will be useful for investigations of ribozyme-substrate interactions.  2000 Elsevier Science B.V. All rights reserved. Keywords: Surface plasmon resonance; Hammerhead ribozymes; RNA substrates; Dynamic interactions

*Corresponding author. Tel.: 1 46-8-5858-3724; fax: 1 46-8-779-5383. ¨ E-mail address: [email protected] (L. Ahrlund-Richter) 1 Both authors contributed equally to this article. 0165-022X / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0165-022X( 99 )00058-5

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1. Introduction Ribozymes are a class of ribonucleic acid (RNA) molecules that possess enzymatic properties. Upon binding to complementary nucleic acid strands and in the presence of certain divalent cations, cleavage of the target RNA takes place. The hammerhead ribozyme is a small ribozyme found in the plus strand of the satellite RNA of tobacco ringspot virus [1]. The use of catalytic RNAs in the study of gene function has attracted great interest and ribozyme inhibition of gene expression is a rapidly developing field (reviewed in Ref. [2]). Ribozymes have also been considered as therapeutic agents that could be used to combat infectious diseases, particularly viruses, where adaptive immunity, chemotherapy or immunisation are not sufficient to eliminate infected cells [3,4]. To date, the in vitro analysis of ribozyme–RNA substrate kinetics has primarily relied on indirect detection methods typically using radioactively labelled RNA and electrophoretic separation techniques. The major drawback of this approach is that the kinetics, at best, can be measured on the minute time scale rather than in real-time. The recent employment of fluorescent resonance energy transfer (FRET) measurements in the study of ribozyme–substrate kinetics has made it possible to follow the kinetic pathway in real-time [5]. An additional alternative is offered by the surface plasmon resonance (SPR) biosensor methodology, which allows molecular interactions to be monitored in real-time and in a dynamic flow without any separation steps for the interactants (reviewed in Refs. [6–8]). Using immobilised target molecules on a dextran matrix and the respective substrate molecules in solution, successive phases of interactions can be monitored. Furthermore, a high sensitivity allows for molecules in the picomolar concentration range to be readily detected. To this date the applications of the SPR technique have primarily been used for the analysis of protein–protein interactions and to some extent the study of protein–DNA interactions [6,8–12] or DNA–DNA interactions [10,13–15]. A few RNA–RNA studies have recently emerged, including one describing a minimised ribozyme cleavage of a substrate [16]. In this present study we have investigated the potential use of the SPR technology to follow the progress of binding, dissociation and cleavage events in a hammerhead ribozyme–substrate model system. We found that specific RNA-binding and dissociation from a non-cleavable substrate could be monitored in real-time utilising the SPR technology. We also found that ribozyme-mediated cleavage of a cleavable substrate could be measured.

2. Materials and methods

2.1. The hammerhead ribozyme–substrate model system The in vitro transcribed and purified RNAs used in this study (S-Rz, specific ribozyme; C-Rz, control ribozyme; S-Sub, cleavable substrate; and mut S-Sub, noncleavable substrate) are schematically outlined in Figs. 1 and 2. In addition, yeast tRNA (Boerhinger Mannheim) was used as control for non-specific RNA–RNA binding (Fig.

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Fig. 1. Schematic illustration of the ribozymes and substrates used in this study (see text). Hypothetical Watson–Crick base pairing (bold lines) and non-Watson–Crick base pairing (bold dots) within the target binding domains are indicated.

2A). The binding domain of S-Rz is fully complementary to the substrates used (Figs. 1 and 2C), while the target-binding domain of C-Rz is 40% homologous to that of S-Rz (Figs. 1 and 2B). S-Rz was derived from the hammerhead ribozyme Rzb [17] which was designed to bind and cleave a 16-ribonucleotide sequence GAGAUGUCAGAUAUGU (the cleavage motif GUC at position 3127–3129 in exon II of the mouse b 2 -microglobulin gene, b2M). C-Rz was derived from the hammerhead ribozyme hhRznef9016 – 9029 [18], designed to bind and cleave a 14-ribonucleotide sequence UUCCAGUCAGACCU (position 9016–9029 in the HIV-1 SF2 genome). As seen in Fig. 1, C-Rz shows potential binding to the S-Rz specific target site in the substrates, via totally nine ribonucleotides (of which three form non-Watson–Crick base pairs). The two substrates used in this study, S-Sub and mut S-Sub, both include the full S-Rz target sequence flanked by two AU-hairpin loops (Fig. 2). The flanking hairpin loops were added in order to increase the size and thus the response levels registered in the sensorgrams. Due to the strategy chosen for immobilisation of the substrates via three biotinylated cytidines at the 39-end (below), there was a need to avoid the use of target sequences containing cytidine residues. The wild-type hammerhead ribozyme target motif GUC was therefore changed to GUA in S-Sub, that has been reported to have a cleavage efficacy of between 30 and 104% of that of GUC [19]. The corresponding triplet in mut S-Sub was mutated to GUG, which has been reported to be non-cleavable [20].

2.2. DNA templates DNA templates for in vitro transcription of the S-Rz and C-Rz ribozymes were

44 T. Nyholm et al. / J. Biochem. Biophys. Methods 44 (2000) 41 – 57 Fig. 2. Predicted interactions between yeast tRNA, C-Rz, S-Rz and immobilised substrates. (A) No binding of tRNA to either mut S-Sub (GUG) or S-Sub (GUA). (B) Partial binding and dissociation of C-Rz from mut S-Sub and S-Sub, but no cleavage of S-Sub. (C) Binding and dissociation of S-Rz from mut S-Sub and S-Sub. (D) Active complex formation between S-Rz and S-Sub, leading to cleavage of S-Sub and subsequent dissociation of the 59-cleavage product and S-Rz from the sensor surface. The three biotinylated cytidine residues at the 39-end of S-Sub and mut S-Sub are indicated by the lower case letter b.

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generated from previously characterised plasmid constructs, Rzb [17] and hhRznef9016 – 9029 [18]. In order to generate in vitro transcripts identical to the ones previously shown to be functional in eukaryotic cells, the CMV promoter was replaced with a T3 RNA polymerase promoter by PCR using the 59 primer AAT TAA CCC TCA CTA AAG GGT CAG ATC GCC TGG TAA TAC GAC. The 39 primers used were 59-GAC GGA TCA GAT CT-39 (S-Rz) and 59-GAC GGA TCA GAT CTG TTC CAG TTT-39 (C-Rz). The PCR products were then cloned into SmaI-digested pUC19. The plasmid templates were finally linearised with KpnI and blunt-ended with Klenow before they were used for in vitro transcription. ssDNA templates for in vitro transcription of the substrates were generated from synthesised oligos (kind gift from Pharmacia Biotech). The top strand with T7 RNA polymerase promoter element 59-AAT TTA ATA CGA CTC ACT ATA G-39 was annealed with either the template strand for S-Sub 59-GGG AAT TAA TTA ATT CCC CCC AAT TAA TTA ATT ACA TAT CTT ACA ]] TCT CTA CCC AAT ATA TAT AAC CCC CCT TAT ATA TAT TCC CTA TAG TGA GTC GTA TTA AAT T-39 (cleavage triplet underlined; GUA in RNA) or with the template strand for mut S-Sub, which is identical to that for S-Sub except for the cleavage triplet (CAC 5 GUG in RNA).

2.3. In vitro transcriptions and purification of RNA Ribozymes were transcribed from the linearised plasmid DNA templates described above following the protocol supplied in the T3 Maxi-script kitE (Promega). 39Biotinylated S-Sub and mut S-Sub were synthesised using T7 MEGAshortscript kitE (Ambion) following the protocol supplied with the 1 mM biotin-14-CTP (Life Technologies). The substrates produced in this way contained three biotinylated cytidine residues at the 39-end. After DNaseI-treatment, all transcripts were purified on 8% Sanger polyacrylamide 8 M urea gels and full-length RNAs were located by UVshadowing at 253 nm on fluorescent TLC plates (Sigma). The excised bands were eluted in 0.5 M NH 4 Ac, 0.2% SDS and 1 mM EDTA (solution F, RPA II-kit, Ambion) for 4 h at 378C. Gel fragments were removed by filtration of the eluates through 0.2-mm filters. The ribozyme transcripts were then transferred to Tris buffer (10 mM Tris, pH 8.0, plus 50 mM spermidine) and the substrates to HBS buffer (10 mM Hepes, pH 7.4, 0.15 M NaCl, 3.4 mM EDTA and surfactant P20, BIAcore AB, Sweden) by ultrafiltration in Centricon-30 tubes (Amicon) at 48C. Concentrations were determined spectrophotometrically at 260 nm, using the following extinction coefficients (C-Rz and S-Rz: 1.05 3 10 6 M 21 cm 21 ; and S-Sub and mut S-Sub: 9.25 3 10 5 M 21 cm 21 ). The purity of the ribozyme and substrate preparations were finally confirmed on 6 or 8% Sanger polyacrylamide 8 M urea gels by ethidiumbromide staining.

2.4. Preparation of 32

32

P-labelled RNA substrates

P-labelled substrates were generated following the T7 MEGAshortscriptE protocol (Ambion) with 10 mCi [a- 32 P]UTP present plus all NTPs at a final concentration of 0.75 mM. After DNaseI-treatment, the full-length RNAs were identified on an 8% Sanger

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polyacrylamide 8 M urea gel by autoradiography, excised and eluted from the gel as above for 1 h. Finally, the RNA eluates were transferred to 10 mM Tris, pH 8.0, containing 50 mM spermidine using NICK-columns (Pharmacia Biotech). The purity of the 32 P-labelled substrate preparations were checked on a 8% Sanger polyacrylamide 8 M urea gel by autoradiography.

2.5. Gel analysis of cleavage with

32

P-labelled RNA substrates

Ribozyme cleavage reactions were performed at 378C in 15 ml with the same buffer used for the SPR measurements (see below). The C-Rz (106-mer), S-Rz (107-mer) and the S-Sub and mut S-Sub substrates (both 86-mer) were separately heat-denatured in 10 mM Tris, pH 8.0, plus 50 mM spermidine at 908C for 1 min, and cooled to the reaction temperature. The reactions were initiated by mixing the ribozyme and substrate components in a 1:1 molar ratio and adding MgCl 2 to a final concentration of 2 mM. Two-ml aliquots were removed after 10 min and 1 h and the reaction was stopped with 2 vol of gel loading buffer containing 80% formamide plus 50 mM EDTA and stored at 2 208C until analysis. Substrates and cleavage products were separated on 8% Sanger polyacrylamide 8 M urea gels, autoradiographed and imaged on a FUJIX Bio-Image Analyser and measured using the BAS 2000 software.

2.6. Surface plasmon resonance measurements Real-time analysis of ribozyme and substrate interactions were performed on a BIAcore2000E instrument (BIAcore AB, Uppsala, Sweden), which allows for simultaneous measurements in four flow-cells. The detection principle of SPR is based on the refractive index changes in the optical SPR signal resulting from changes in mass concentration at the sensor surface in a flow cell [21]. The sensor surface consists of a carboxymethylated dextran layer, which is attached to a thin gold film. On the other side of the gold film monochromatic light interacts with free oscillating electrons (plasmons) in the gold film and is internally reflected through a glass prism. When immobilisation of one molecular species in the dextran layer and subsequent interactions with a non-immobilised molecular species are performed under continuous laminar flow over the sensor surface, the intensity of the reflected light changes. The changes in light intensity give rise to the SPR signal, which is proportionally related to the change in mass concentration at the sensor surface. The mass-related change in the SPR signal is displayed in resonance units (RU) and plotted against time in a sensorgram, where 1 RU 5 1 pg bound nucleic acids mm 22 , which approximates that for proteins [22,23].

2.7. Immobilisation of biotinylated RNA substrates The 39-biotinylated substrates (S-Sub and mut S-Sub) were immobilised on individual SA5 sensor chips (BIAcore AB, Uppsala, Sweden), which contained approximately 2400 RU of streptavidin. For this procedure, 6 ml of low, medium and high amounts of the substrates (125 fmol, 380 fmol and 5.5 pmol) were injected separately over three

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flow-cell surfaces at a flow-rate of 3 ml / min at 378C, leaving one flow-cell as a reference. This resulted in the following mean immobilisation responses: 163625 RU (LD, low density), 489618 RU (MD, medium density) and 1147657 RU (HD, high density) for mut S-Sub, and, 317613 RU (LD), 673611 RU (MD) and 1089617 RU (HD) for S-Sub. Each immobilisation procedure was followed by one 5-ml injection of a regeneration solution (4 M urea plus 15 mM EDTA) to remove non-immobilised substrate and to assess the stability of the streptavidin–biotin–substrate coupled complexes.

2.8. Ribozyme-RNA substrate assays on BIAcore2000E All experiments were performed at 378C, at a flow-rate of 2 ml / min in a buffer containing 10 mM Tris, pH 8.0, 2 mM MgCl 2 plus 50 mM spermidine. The same buffer was used as running buffer continuously flowing over the sensor surface. All solutions (ribozyme samples, running buffer and regeneration solution) were degassed prior to the experiments by vacuum. MgCl 2 was added to yeast tRNA, C-Rz and S-Rz samples to a final concentration of 2 mM prior to the SPR measurements. tRNA or ribozyme samples were injected in a concentration series of 25, 50, 100 and 200 nM in 300-s pulses over sensor surfaces with immobilised mut S-Sub (LD, MD and HD). First tRNA, then C-Rz followed by S-Rz were sequentially injected three times at each concentration. After the injections, dissociation of bound ribozymes was followed during 800 s under continuous flow of running buffer. After each ribozyme sample measurement, the substrate-matrices were regenerated with a 5-ml pulse of 4 M urea plus 15 mM EDTA and equilibrated with the running buffer. In the subsequent cleavage experiments, the highest concentration (200 nM) of tRNA and the ribozymes was selected to be injected over the sensor surfaces with S-Sub immobilised at different densities (LD, MD and HD) following the experimental procedure described above, except that the dissociation was followed for 1400 s (400 s for tRNA). The RU values were recorded at 1-s intervals and collected raw data were processed with the BIAevaluation 3.0 software (BIAcore AB, Uppsala, Sweden).

3. Results

3.1. Assay of ribozyme mediated cleavage using

32

P-labelled RNA substrates

Prior to the SPR measurements, functional analyses on the cleavage properties of S-Rz (specific ribozyme) and C-Rz (control ribozyme) were performed with 32 P-labelled S-Sub (cleavable substrate) and mut S-Sub (non-cleavable substrate) and detected using gel electrophoresis. Fig. 3 shows that S-Sub was cleaved by S-Rz at equimolar amounts, with a cleavage efficacy of 67% obtained after 10 min (lane 6). Extending the reaction to 1 h resulted in an additional cleavage of 14% (lane 7). The expected substrate cleavage products, a 44-mer 39-fragment and a 42-mer 59-fragment are indicated in the autoradiogram. C-Rz did not display any cleavage activity against S-Sub (lanes 4 and 5). No cleavage of mut S-Sub was detected in the presence of S-Rz or C-Rz (data not shown)

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Fig. 3. Autoradiogram illustrating specific and increasing cleavage of S-Sub after 10 and 60 min incubation with S-Rz. Mw-marker (10-bp DNA ladder) (lane 1). S-Sub incubated for 10 min (lane 2) or for 60 min (lane 3) without any ribozyme present. S-Sub incubated with C-Rz for 10 min (lane 4) or for 60 min (lane 5). S-Sub incubated with S-Rz for 10 min (lane 6) or 60 min (lane 7).

even after 1 h. Nor was any background cleavage detected after incubation without ribozyme (lanes 2 and 3).

3.2. Immobilisation and stability of biotinylated RNA substrates In order to test the capacity of the BIAcore instrument to measure RNA–RNA interactions, we first immobilised different concentrations of 39-biotinylated mut S-Sub and S-Sub substrates to streptavidin-coated sensor chips. Injections of different amounts (125 fmol, 380 fmol and 5.5 pmol) of mut S-Sub and S-Sub resulted in an instantaneous increase in the responses detected by the BIAcore instrument. These reached the final mean immobilisation levels of 163625 RU (1262 fmol / mm 2 ; LD), 489618 RU (3661 fmol / mm 2 ; MD) and 1147657 RU (7864 fmol / mm 2 ; HD) for mut S-Sub as shown in Fig. 4A. For comparison, the corresponding immobilisation levels obtained for S-Sub were 317613 RU (2361 fmol / mm 2 , LD), 673611 RU (4961 fmol / mm 2 , MD) and 1089617 RU (7661 fmol / mm 2 , HD). To assess the stability of the streptavidin–biotin– RNA coupled complex against the regeneration procedure, the RNA-matrices were subjected to one injection of regeneration solution (4 M urea plus 15 mM EDTA) before ribozyme sample injections. As shown in Fig. 4A, the mut S-Sub–biotin–streptavidin complex was found to be intact after one 5-ml pulse of regeneration solution. The observed drift for both substrates, 2 1967 RU (LD), 2 28613 RU (MD) and

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Fig. 4. (A) Sensorgram showing the immobilisation and stability of mut S-Sub on three matrix surface sites on the sensor chip (not corrected for bulk response (BR)). The following phases are indicated: (a) initial baseline response to the running buffer passing over the surface. Injection of biotinylated mut S-Sub (125 fmol, 380 fmol and 5.5 pmol) over the streptavidin-modified surfaces for 120 s (arrows) with the corresponding increase of the baseline (b) following the immobilisation at three different levels of mut S-Sub, 147 RU (LD), 472 RU (MD) and 1108 RU (HD). (c) Washing of the mut S-Sub-coated matrix with one injection of 5 ml of regeneration solution (4 M urea plus 15 mM EDTA) for removal of non-immobilised mut S-Sub molecules. (B) Example sensorgram showing the injection of S-Rz (not corrected for BR) over one of the mut S-Sub-coated matrix surfaces ( mut S-Sub HD) and the same injection over the empty reference cell surface. The following phases are indicated: (a) a sample of S-Rz (200nM) was injected over the surface in a pulse of 300 s (arrows). The increasing response during sample injection is the result from net association between S-Rz and immobilised mut S-Sub. (b) Post-injection, the net dissociation of bound S-Rz molecules is registered in the sensorgram for 800 s. Non-dissociated S-Rz molecules are subsequently removed from the matrix (c) with a 5-ml pulse of the regeneration solution, after which the baseline returns to the response level of immobilised mut S-Sub.

2 20610 RU (HD), correspond to 864, 562 and 261% of the respective immobilisation responses taken immediately before the regeneration pulse. This drift was mainly attributed to removal of non-immobilised substrate molecules since the baseline remained fairly stable over long time runs on one and the same chip. Only a small drift of 2 1.160.3 RU or 0.7% (LD), 2 3.861.9 RU or 0.8% (MD) and 2 4.361.3 RU or

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Fig. 4. (continued)

0.4% (HD) was observed ( mut S-Sub) following the treatment of each injected ribozyme or tRNA sample with regeneration solution. In addition to this ‘urea-effect’, a minor baseline drift of 2 0.0006 RU / s (LD), 2 0.001 RU / s (MD) and 2 0.003 RU / s (HD) over the background drift observed in the reference cell was measured between the sample injections.

3.3. Real-time interactions between S-Rz, C-Rz and

mut

S-Sub

The ability of S-Rz and C-Rz to bind to the immobilised mut S-Sub and S-Sub substrates was evaluated on the BIAcore instrument. In a typical experimental cycle (Fig. 4B) a S-Rz sample was injected over a sensor surface (a) on which non-cleavable mut S-Sub had been immobilised. Bound S-Rz molecules dissociated from the mut S-Sub matrix surface under continuous flow of running buffer (b) and non-dissociated S-Rz was removed by regeneration of the mut S-Sub matrix (c). A bulk response (BR) was seen during all sample injections due to differences in composition of the running buffer and sample buffer [24]. The BR was corrected for each sample injection by subtracting the

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responses obtained from corresponding sample injection over an empty surface (reference cell). In this way, baseline drift and non-specific binding of samples to the dextran matrix was also detected and corrected for. As illustrated with S-Rz in Fig. 4B, the injected samples displayed very low or no affinity for the dextran matrix. The results from multiple experiments using ribozymes and tRNA on mut S-Sub at different immobilisation levels (147 RU (LD), 472 RU (MD) and 1108 RU (HD)) are shown in Fig. 5A–C. The superimposed curves describe the relative binding and dissociation profiles for C-Rz (Fig. 5A) and S-Rz (Fig. 5B) followed in real-time in a concentration series ranging from 25 to 200 nM. It can been seen that S-Rz binds to mut S-Sub with higher affinity than C-Rz. The lower affinity of C-Rz for mut S-Sub is also illustrated by the faster dissociation from mut S-Sub during the post-injection time (300–1100 s). Under the experimental conditions used, S-Rz failed to fully saturate the binding sites on the mut S-Sub-matrices within the injection time (with a maximum of 5260.6% of mut S-Sub (LD) complexed with S-Rz at the highest concentration (200

Fig. 5. (A) Overlaid sensorgram plots for interaction between C-Rz and mut S-Sub, immobilised at three different levels: 147 RU (LD), 472 RU (MD) and 1108 RU (HD). The superimposed curves (corrected for BR) show sequential injections of C-Rz in a concentration series of 25 nM (bottom curve), 50 nM (lower intermediate curve), 100 nM (higher intermediate curve) and 200 nM (top curve). (B) Overlaid sensorgram plots for interaction between S-Rz and mut S-Sub, immobilised at three different levels: 147 RU (LD), 472 RU (MD) and 1108 RU (HD). The order of the superimposed curves (corrected for BR) is the same as in (A). (C) Non-specific binding between tRNA and mut S-Sub, immobilised at three different levels: 147 RU (LD), 472 RU (MD) and 1108 RU (HD). The order of the superimposed curves (corrected for BR) is the same as in (A).

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nM)). In the case of C-Rz, a maximum saturation of 3661.4% of mut S-Sub (LD) was obtained. The percentage ribozyme saturation of mut S-Sub binding sites was calculated by dividing the binding responses for S-Rz and C-Rz at 100 s post-injection time by the theoretical value of the maximum binding capacity (R max ) of immobilised mut S-Sub (RU /R max 3 100 5 x% saturation), assuming a 1:1 stoichiometry between the ribozyme and substrate. The R max was calculated by multiplying the ribozyme to mut S-Sub molecular weight ratio (1.12:1) by the amount of immobilised mut S-Sub (147 RU, 149 RU or 192 RU (LD), 472 RU, 489 RU or 507 RU (MD) and 1108 RU, 1121 RU or 1212 RU (HD)) giving an R max for each surface of 163 RU, 167 RU or 215 RU (LD), 543 RU, 548 RU or 568 RU (MD) and 1230 RU, 1256 RU or 1357 RU (HD). The degree of saturation gradually decreased for both S-Rz and C-Rz at higher immobilisation levels of mut S-Sub (MD, 3960.9 and 2761.4%; HD, 1960.5 and 1360.5% for 200 nM of S-Rz and C-Rz, respectively). When tRNA was tested, no significant binding to the immobilised mut S-Sub could be detected even at the highest concentration tested (200 nM, Fig. 5C).

3.4. SPR measurements of S-Rz cleavage of S-Sub Specific cleavage of S-Sub by S-Rz and C-Rz ribozymes was evaluated in several independent experiments. For each experiment three consecutive injections of C-Rz or tRNA were followed by three consecutive injections of S-Rz (all at 200 nM) over streptavidin-coated dextran matrices onto which S-Sub had been immobilised at different levels: 317613 RU (LD), 673611 RU (MD) and 1089617 RU (HD). One representative immobilisation level (669 RU, MD) is shown in Fig. 6A–C. Injections of C-Rz (Fig. 6A) resulted in lower binding responses with S-Sub (S-Sub 5 669 RU), compared to immobilised mut S-Sub ( mut S-Sub, 472 RU; see ‘200 nM-curve’ in Fig. 5A), and, faster dissociation from S-Sub as shown by the three sequential injection curves superimposed on each other. After pulsing with regeneration solution the baseline returned to a level slightly lower than the level before S-Sub was challenged with C-Rz. The total drop in the S-Sub baseline after three C-Rz injections was 2 2763 RU, 2 2866 RU and (no data at HD) at respective S-Sub surfaces. Fig. 6B shows three sequential injections of S-Rz over the surface with S-Sub immobilised at the medium density (669 RU). Interactions of S-Rz with S-Sub resulted in slightly lower binding responses compared to its interactions with mut S-Sub (see ‘200 nM-curve’ in Fig. 5B), followed by a faster lowering of the sensorgram curves during the dissociation phase. Subsequent regeneration after the first injection, resulted in a mean drop of the responses to 2 3561, 2 5866 and 2 72616 RU below the baseline at time 0 s (0 RU) for S-Sub (LD, MD and HD, respectively). Subsequent second and third injections of S-Rz over the different S-Sub surfaces, resulted in gradually decreased binding (Figs. 6B and 7A) and postregeneration responses (Figs. 6B and 7B) with an accumulative lowering of S-Sub baseline of 2 7667, 2 142645 and 2 17061 RU for LD, MD and HD, respectively. When testing tRNA, no or very low non-specific binding to S-Sub could be detected, as shown in Figs. 6C and 7A. A drop in the S-Sub baseline was observed after each regeneration procedure, but since tRNA displayed very low affinity for S-Sub, these responses should reflect loss of S-Sub from the surface due to the regeneration procedure

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Fig. 6. (A) Overlaid sensorgram plots for interaction between C-Rz and S-Sub, immobilised at 669 RU (MD). The superimposed curves (corrected for BR) show serial injections of C-Rz (200 nM) for 300 s, post-injection interaction for 1400 s and regeneration of the surface. (B) Overlaid sensorgram plots (corrected for BR) for the S-Rz-mediated cleavage of S-Sub, immobilised at 669 RU (MD). The order of the serial injections of S-Rz (200 nM) is indicated by 1 (first injection), 2 (second injection) and 3 (third injection). The time windows for the different measurement stages are the same as for C-Rz. (*) Cleavage is illustrated in quantitative terms by comparing the change of the S-Sub baselines after injection of the regeneration solution. (C) Overlaid sensorgram plots for non-specific interaction between tRNA and S-Sub, immobilised at 669 RU (MD). The superimposed curves (corrected for BR) show serial injections of tRNA (200 nM).

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Fig. 7. (A) Diagram showing the binding responses of tRNA, C-Rz and S-Rz to immobilised S-Sub. The binding responses (at 100 s post-injection time) for tRNA, C-Rz and S-Rz was obtained from several injections (200 nM) on the different S-Sub matrices (LD, MD and HD). Data for C-Rz (HD) is missing. (B) Diagram illustrating the percentage (%) remaining S-Sub after the binding (and cleavage when applicable) of the tRNA, C-Rz and S-Rz, respectively, to the immobilised S-Sub. In order to determine percentage (%) cleavage, the initial amount of cleavable S-Sub (x RU) was calculated from the relative contribution of the 59-fragment (non-biotinylated) to the individual immobilisation levels; 313 or 332 RU (LD), 669 or 685 RU (MD) and 1070 or 1104 RU (HD). This was done by dividing the relative molecular weight of the 59-fragment (1) with that of the sum of the 59-fragment (1) and 39-fragment (1.11) multiplied by the RU value for each immobilisation level ( y), giving (1 /(1 1 1.11))y RU 5 x RU. This resulted in an initial amount of ‘cleavable’ S-Sub (non-biotinylated 59-fragment) of 145 or 157 RU (LD), 317 or 325 RU (MD) and 507 or 523 RU (HD). The cleavage responses for S-Rz were normalised to the post-regeneration responses obtained from the tRNA and C-Rz injections (see text for explanation).

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rather than tRNA-mediated cleavage. As the post-regeneration responses for C-Rz were comparable to that of tRNA, also C-Rz was considered to lack cleavage activity against S-Sub. Hence, to obtain the actual cleavage responses for S-Rz, all post-regeneration responses for S-Rz were normalised to the corresponding responses observed in the tRNA- (and C-Rz) -S-Sub experiments: 2 762 RU or 2.160.5% (LD), 2 1161 RU or 1.760.1% (MD) and 2 963 RU or 0.860.3% (HD). The corrected cleavage responses for S-Rz were then divided by the initial amount of ‘cleavable’ S-Sub (non-biotinylated 59-fragment; see legend in Fig. 7B for calculations) of 145 or 157 RU (LD), 317 or 325 RU (MD) and 507 or 523 RU (HD) to obtain the percentage S-Sub remaining after cleavage (Fig. 7B). The first injection of S-Rz reduced the amount of S-Sub to 79.561.3% (LD), 83.761.5% (MD) and 86.462.9% (HD) of the preceding values. The corresponding values for the subsequent second and third injections of S-Rz were calculated in the same way, by subtracting the cleavage responses from the amount of ‘cleavable’ S-Sub remaining after the preceding injection. Hence, further cleavage following the second injection reduced the amount of cleavable S-Sub to 63.860.2% (LD), 69.865.8% (MD) and 76.762.2% (HD), and to 52.362.5% (LD), 57.8613.0% (MD) and 67.460.6% (HD) after the third injection.

4. Discussion The results in this study suggest that the SPR methodology is well suited for characterising interactions between hammerhead ribozymes and RNA substrates for the following reasons. First, the main advantage of using SPR, is that ribozyme–substrate interactions can be monitored without use of label in the hybridising sequences, such as fluorophores in FRET, which have been shown to interfere with the specific interactions [25]. In SPR, the only requirement is an end-label for anchoring the substrates to the sensor surfaces. The biotin end-label used here has previously been used in DNA–DNA hybridisation experiments without interfering with the specific interactions [15]. Capturing one of the RNA interactants also makes the immobilised RNA reusable for subsequent interactions. Due to this and to the high sensitivity of SPR, relatively small amounts of the RNA interactants are needed, approximately 1000-fold less than required for FRET [5]. In addition a simultaneous analysis of multiple cells in parallel, with different amounts of immobilised RNA and a blank reference surface, makes it is possible to distinguish the reaction of interest from mass-transport effects and instrumental artefacts such as baseline drift, non-specific binding to the dextran matrix and bulk response. Secondly, reproducible immobilisation levels and efficient anchoring of biotinylated substrates were obtained by taking advantage of the strong coupling chemistry of biotin and streptavidin [26]. The binding capacity of the immobilised substrate-matrices were unaffected by continuous running up to 14 h on one and the same chip. A baseline drift slightly higher than the maximum instrumental drift reported for BIAcore2000 was observed in some experiments [27]. The identity of this effect is unknown but may be explained by a small non-specific degradation of the substrates. The drift may also originate from a slow continuous leakage of biotinylated RNAs and breakage of the

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biotin–streptavidin non-covalent interaction caused by the injections of regeneration solution. The biotin–streptavidin complex has, however, been reported to be intact after extended incubations at higher urea concentrations (6 M) [26] than used here (4 M). Another explanation may be a non-specific RNA-degradation due to contamination by RNase, even though precautions were taken by using RNAse-free reagents. Furthermore, all material used were thoroughly autoclaved and the SPR instrument was treated with sodium hydroxide and urea prior to each experiment. Due to the strong interaction forces between biotin and streptavidin there is no direct method for removal of biotinylated RNA to reuse the chip surfaces, without destroying the matrix. Consequently a new streptavidin-coated chip, with comparable amounts of immobilised streptavidin, was used in each experiment. Thirdly, the results showed that qualitative ranking of the ribozymes in terms of affinity was possible simply by visual comparison of the appearance of sensorgrams. As predicted, the interactions between the S-Rz ribozyme and the complementary noncleavable substrate ( mut S-Sub) revealed a stronger binding, higher amount of complexes formed and a slower decay of the complexes than for the less complementary C-Rz. The interactions were specific, indicated by the near absence of ribozymes binding to the dextran matrix in the reference cell, and by the very low non-specific binding to immobilised substrate by tRNA. The surface concentration of mut S-Sub does not seem to be a limiting factor for the binding of C-Rz ribozyme when compared to S-Rz (Fig. 5A,B). While the binding curves for S-Rz display an increased binding, the curves for C-Rz are almost superimposable at medium and high density (MD and HD) mut S-Sub. These differences in binding behaviour may be explained by a lower melting temperature of the C-Rz– mut S-Sub complex as compared to S-Rz– mut S-Sub complex. Finally, ribozyme cleavage was readily detected by SPR. A significant lowering of the S-Sub baseline was seen after the first injection. After removal of non-dissociated S-Rz and 59-cleavage products with regeneration solution, the specific cleavage of S-Sub was 20- to 35-fold above the non-specific background seen with mut S-Sub. The lowered binding and cleavage responses, observed from the subsequent injections of S-Rz (Figs. 6B, 7A,B) over cleaved S-Sub, also indicate that the amount of S-Sub was reduced stepwise by S-Rz cleavage. In contrast, no indication of cleavage by C-Rz was found in the gel analysis or by SPR. A minor non-specific lowering of the S-Sub baseline in the presence of C-Rz and tRNA, was comparable to that of the non-cleavable mut S-Sub.

5. Simplified description of the method and its future applications Specific binding to and dissociation from a non-cleavable RNA substrate by ribozymes could readily be followed in real-time using the SPR biosensor technology. Also specific ribozyme-mediated cleavage of a cleavable substrate could be clearly detected. In the light of these results the application of SPR technology could be very useful for investigating fundamental aspects of RNA–RNA kinetics such as binding, dissociation and cleavage processes.

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Acknowledgements This work was supported by grants from The Swedish Medical Research Council, Funds at the Karolinska Institutet. We are also grateful for the support of this investigation by BIAcore AB and for kindly providing us the BIAcore2000 instrument to perform all experiments.

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