Cycling probe technique

Research Reports

F. Bekkaoui, I. Poisson1, W. Crosby1, L. Cloney and P. DuckID Biomedical, Burnaby, BC,and 1National Research Councilof Canada, Saskatoon, SK,Canada

ABSTRACT

A streptavidin-RNase H gene fusion wasconstructed by cloning the Thermus ther-mophilus RNase H coding sequence in thestreptavidin expression vector pTSA18F.The gene was expressed in Escherichia coli,and the resulting fusion protein was puri-fied to apparent homogeneity. The fusionprotein was shown to have a molecularweight of 128 kDa and to consist of foursubunits. Furthermore, heat treatment ofthe fusion enzyme showed that it was stableas a tetramer at 65C. The fusion enzymewas shown to have both biotin binding andRNase H catalytic properties. Using cyclingprobe technology (CPT), the fusion enzymewas compared to the native RNase H with abiotinylated probe at different ratios ofprobe:enzyme and varying amounts of syn-thetic target DNA. At a ratio of 1:1, the fu-sion enzyme was active in CPT, but the na-tive enzyme was not; both enzymes wereactive at a 1:5000 ratio of probe:enzyme.The fusion enzyme was further tested usingbiotinylated and non-biotinylated probesand was shown to be active at a 1:1 ratiowith the biotinylated probe but not with thenon-biotinylated probe. These experimentsshow that through binding of the strepta-vidin-RNase H fusion enzyme to the biotiny-lated probe, the efficiency of the cyclingprobe reaction is enhanced.

INTRODUCTION

RNase H is an enzyme that spe-cifically degrades the RNA portion ofDNA/RNA hybrids (3,8). The enzymedoes not cleave single-stranded DNAor RNA. This property has allowed thedevelopment of cycling probe technol-ogy (CPT), a method for detection andquantification of low amounts of targetDNA (5,7). The principle of this meth-od, based on RNase H activity and theuse of chimeric probes DNA-RNA-DNA, is outlined in Figure 1. The reac-tion is carried out at a temperature thatallows the chimeric probe to anneal tothe single-stranded target DNA. RNaseH cuts within the RNA portion of thechimeric probe, and the shorter cleavedprobe fragments dissociate from thetarget, thereby regenerating the targetfor further cycling. The resulting accu-mulation of probe fragments can sub-sequently be detected. CPT has poten-tial applications in infectious andgenetic disease diagnostics. Since thetarget DNA is not amplified, CPT ex-hibits low background. Furthermore,CPT is fast, linear, isothermal and sim-ple compared with other DNA detec-tion methods.

High concentrations of RNase H arerequired for the detection of lowamounts of target DNA. Attachment ofthe RNase H to nucleic acid is an ap-proach to reduce the amount of enzymeused and to increase the efficiency andspecificity of RNase H in CPT. An en-zyme conjugate has been previouslyused to increase the efficiency andspecificity of nucleic acid enzymes bycovalently attaching an oligodeoxyri-bonucleotide to a ribonuclease (2). Asimilar approach was used to link ananodeoxyribonucleotide to RNase H(11).

Another strategy to improve the eff-iciency and the specificity of RNase isto generate a fusion protein betweenRNase H and streptavidin. A biotinyl-ated nucleic acid could then be boundto this fusion enzyme. Since CPT isgenerally performed at 65C for in-creased specificity, the thermostableRNase H from Thermus thermophilus(9) was chosen. In this paper, we de-scribe the cloning, purification and thecharacterization of a streptavidin-RNase H fusion protein. The resultsshow that this fusion protein is active inCPT experiments at very low concen-trations when bound to a biotinylatedprobe. This novel enzyme can be usedfor detection of low amounts of DNAusing CPT, or for the cleavage of RNAin antisense studies.

MATERIALS AND METHODS

Cloning of the Streptavidin-RNaseH Gene Fusion

General cloning techniques were asdescribed by Sambrook et al. (16). Un-less otherwise indicated, modifying en-zymes were from Life Technologies(Gaithersburg, MD, USA). DNA se-quencing was performed by thedideoxy termination method (17). TheRNase H gene from T. thermophilus (9)was polymerase chain reaction (PCR)-amplified from genomic DNA usingprimers BC202 5-CCG AAT TCTTAT GCC TCT TCG TGA-3 andBC203 5-CCG AAT TCA ACC CCTCCC CCA GGA-3. The amplifiedproduct was cloned as a blunt-endedfragment into the SmaI site of pTZ19R(Pharmacia Biotech, Montreal, QC,Canada) resulting in plasmid pIDB1.Primers FB102 5-CCG CAT ATGAAC CCC TCC CCC AGG-3 and BC-202 were used to amplify the corre-

240 BioTechniques Vol 20, No. 2 (1996)

Cycling Probe Technology with RNase HAttached to an Oligonucleotide BioTechniques 20:240-248 (February 1996)

sponding fragment from pIDB1. It wassubsequently cloned as a blunt-endedfragment into the SmaI site of pTZ19R.The resultant plasmid was designatedpIDB6. The restriction fragment NdeI -EcoRI from pIDB6 was then clonedinto pT7-7 (United States Biochemical,Cleveland, OH, USA) to generatepIDB9. This plasmid allows expressionof the RNase H in its native form.

To construct the streptavidin-RNaseH fusion protein, the sequence encod-ing RNase H was PCR-amplified frompIDB1 utilizing primers FB102 andBC202. The amplified fragment wasphosphorylated and ligated into thevector pTSA-18F (18) that had beenpreviously cut with SmaI and treatedwith phosphatase. The resulting plas-mid was designated pIDB10. The vec-tor pTSA-18F contains a DNA se-quence that codes for a truncated formof streptavidin, the expression of whichis controlled by the T7 promoter.

Expression and Purification

Native RNase H was purified as de-scribed (10) with the following modifi-cations. After the second phosphocellu-lose column and concentration of theRNase H fractions, the proteins were

applied to a Superose 12 column(Pharmacia Biotech) that had beenequilibrated with 20 mM sodium ac-etate, pH 5.5, and 150 mM NaCl at aflow rate of 0.4 mL/min. The threefractions corresponding to the RNase Hpeak were pooled and concentratedwith a Centriprep 10 (Amicon, Bever-ly, MA, USA). RNase H was stored at-20C in glycerol buffer (20 mM sodi-um acetate, pH 5.5, 150 mM NaCl,40% glycerol).

Plasmid pIDB10 was transformedinto the bacterial strain NM522 (6). T7RNA polymerase was supplied by in-fecting the recombinant Escherichiacoli with an M13 phage containing theT7 polymerase gene under control ofthe lac UV5 promoter (22,23). NM522cells containing pIDB10 were grown at37C in 1 L of 2YT medium (2YT = 10g yeast extract, 16 g Bacto-tryptone, 5 gNaCl/L, pH 7.0) containing 0.05mg/mL ampicillin. When the culturereached an OD600 of 0.30.4, isopropylb-D-thiogalactopyranoside (IPTG) wasadded to a final concentration of 0.3mM to induce the expression of the T7polymerase gene. After 1 h, 10 mL ofM13 phage (ca. 5 109 plaque-formingunits/mL) were added, and the infectedcells were further grown for 4 h. Cells

Vol. 20, No. 2 (1996) BioTechniques 241

Figure 1. Principle of the CPT reaction. Single-stranded target (I) serves as a catalyst for CPT. In thepresence of excess chimeric probe (DNA-RNA-DNA, the DNA portion is indicated by a straight lineand the RNA portion by a serrated line) (II) and RNase H (III), the RNA portion of the resulting probe-target complex (IV) is cleaved by RNase H at 65C. The shorter cleaved probe fragments dissociate fromthe target, thereby regenerating the target DNA for further cycling (V). The released fragments are re-solved from the uncut probe and detected (VI).

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were harvested by centrifugation at2900 g for 15 min at 4C. Cell pelletswere resuspended in 30 mL of lysisbuffer (1 M Tris-HCl, pH 7.4, 1 mMEDTA) and stored frozen at -70C. Af-ter thawing, the cells were lysed on iceusing a sonicater (Model 450 Sonifier;Branson Ultrasonic, Danbury, CT,USA) and centrifuged at 39 000 g for15 min at 4C. The pellet was resus-pended in 20 mL of urea buffer (20mM sodium acetate, pH 5.5, 8 M urea),homogenized using a 20.5-gaugeneedle and syringe, and incubated over-night at 4C. The sample was againclarified by centrifugation at 39 000 gfor 15 min. The protein solution wasapplied to a 2-mL phosphocellulose(P11; Whatman, Clifton, NJ, USA) col-umn, connected to an FPLC system(Pharmacia Biotech), which had beenequilibrated with urea buffer. The col-umn was washed with 8 M urea, 0.2 MNaCl, and the protein was eluted usinga 0.2 to 0.7 M NaCl linear gradient in 8M urea, 20 mM sodium acetate, pH 5.5.Fractions were pooled and dialyzedovernight, without stirring, in 0.2 Mammonium acetate, pH 6.0, 0.1 mMEDTA and 0.02% NaN3. The samplewas then dialyzed against loadingbuffer (1 M NaCl, 50 mM sodium car-bonate, pH 10.5) for approximately 2 hand centrifuged at 39 000 g for 15min. The supernatant was applied to a2-mL 2-iminobiotin-agarose column(Sigma Chemical, St. Louis, MO,

USA) pre-equilibrated with loadingbuffer. The column was washed withloading buffer, and the protein waseluted with 6 M urea, 50 mM ammoni-um acetate, pH 4.0, and 0.1 mMEDTA. The eluted protein fractionswere pooled and applied to a PD-10 de-salting column (Pharmacia Biotech)equilibrated with 20 mM sodium ac-etate, pH 5.5, and 150 mM NaCl. Pro-tein eluted from the desalting columnwas concentrated with a Centricon-10filter (Amicon) and stored in the sameconditions as the native enzyme. Thepurity of the fusion protein was as-sessed using 12.5% sodium dodecylsulfate polyacrylamide gel electro-phoresis (SDS-PAGE) (13). The con-centration of the fusion protein was de-termined using a spectrophotometer(A280 of 0.1% = 2.3, each subunit withan Mr 32 104).

Oligonucleotide Preparation

Chimeric probes (DNA-RNA-DNA) were synthesized using an auto-mated Applied Biosystems 394 DNA/RNA synthesizer (Perkin-Elmer/ Ap-plied Biosystems Division, Foster City,CA, USA) according to the manufac-turers protocols (ABI User Bulletin#79). The regular 1.0 mmol cyanoethylsynthesis cycle was modified to give a10-min wait step for the coupling reac-tion when ribonucleoside phospho-ramidites were accessed. Base labile

Expedite deoxyribonucleoside andribonucleoside phosphoramidites andExpedite Cap A solution were obtainedfrom Millipore (Bedford, MA, USA).Cold aqueous ammonia solution wasused to cleave the oligonucleotidesfrom the support, to remove the cya-noethyl phosphate protecting groupsand to remove the protecting group onthe bases. Triethylamine trihydrofluo-ride (Aldrich Chemical, Milwaukee,WI, USA) was used to remove the ri-bosilyl protecting group on each 2 hy-droxyl group of the RNA portion of theprobe (21). Two chimeric probes wereused in this study, ARK2, 5-d(GTCGTC AGA CCC) r(AAAA) d(CCCCGA GAG GG)-3, and ARK2B, (bi-otinylated ARK2) 5-d(GTC GTCAGA CCC) r(AAAA) d(CCC CGAGAG GG) S12-B-3, where B is a biotinand S a spacer Phosphoramadite 9(Glen Research, Sterling, VA, USA).The oligonucleotides were purified byC18 reverse-phase HPLC. ARK2T isthe synthetic target DNA complemen-tary to ARK2 and ARK2B.

Streptavidin Binding ActivityStreptavidin binding activity to 32P-

labeled probes was estimated by gelelectrophoresis. The fusion enzymewas incubated with ARK2 or ARK2Bin CPT buffer (8 mM MgCl2 , 50 mMTris-HCl, pH 8.1, 0.025% Triton X-100) containing 1 M NaCl. SDS-PAGEloading buffer (120 mM Tris-HCl, pH


Figure 3. SDS-PAGE analysis of the effect ofheat treatment of the streptavidin-RNase Hfusion protein. Molecular weight standards inkDa (lane 1). Treatment for 20 min in gel loadingbuffer at 25C (lane 2), 37C (lane 3) , 65C (lane4) and 100C (lane 5).

Figure 2. Details of the streptavidin fusion cloning vector pTSA-18F (A) and the streptavidin-RNase H fusion construct (B).

6.8, 4% SDS, 20% glycerol, 0.01%bromophenol blue) was added to theprotein oligonucleotide mixture, and itwas subjected to gel electrophoresis us-ing the PhastSystem (PharmaciaBiotech). Gels were analyzed by aPhosphorImager utilizing Image-Quant software (Molecular Dynam-ics, Sunnyvale, CA, USA ). The per-centage of probe that was bound to thefusion protein was estimated by inte-gration of the bands corresponding tounbound and bound probe.

RNase H Assays

The acid-soluble counts assay wasbased on a previously publishedmethod (4). Briefly, the single-strandedsubstrate M13 phage DNA was tran-scribed using 3H-UTP to create a la-beled RNA/DNA hybrid (12). RNase Henzyme (0.1 0.02 ng) was added to areaction mixture containing 10 mMTris-HCl, pH 9.0, 1 mM MgCl2,0.005% bovine serum albumin (BSA),1 mM b-mercaptoethanol and 13 mMlabeled substrate M13 and then incu-bated for 510 min at 65C. The reac-tion was then stopped by the additionof 50 L of 0.5 mg/mL carrier tRNAplus 150 L of 20% trichloroacetic acid

(TCA) and placed on ice for 510 min.The sample was centrifuged at 14 000g for 5 min at 4C. Supernatant aliquotsof 50 mL were added to 5 mL of liquidscintillant for counting.

Cycling conditions were modifiedfrom a previously published method(5). The two chimeric probes used inthis study were 5-labeled with 32P-ATP (16) using RTG kinase (Pharma-

cia Biotech) and purified by gel elec-trophoresis. Prior to CPT reactions, theenzyme was incubated with the bi-otinylated probe in CPT buffer contain-ing 1 M NaCl. Reactions containing na-tive enzyme and/or non-biotinylatedprobe were used as controls. CPT reac-tions with different ratios of probe:en-zyme (a 1:1 ratio is calculated to con-tain one streptavidin-RNase H fusion

Vol. 20, No. 2 (1996) BioTechniques 243

Figure 4. SDS-PAGE analysis of the binding ofthe fusion streptavidin-RNase H to biotinylat-ed probe ARK2B. Constant amounts of probe (1fmol) were incubated with no streptavidin-RNaseH (lane 1), 1 fmol of streptavidin-RNase H (lane2), 50 fmol of streptavidin-RNase H (lane 3) and5000 fmol of streptavidin-RNase H (lane 4).

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subunit and one probe) were carried outin 20-mL volumes in CPT buffer con-taining 50 mM NaCl for 30 min at65C. The reaction was stopped byadding 20 mL of urea loading buffer (8M urea, 100 mM EDTA, 0.1% bro-mophenol blue, 0.1% xylene cyanol)on ice. The products of CPT were re-solved on a 20% acrylamide, 7 M ureagel at 500 V. The gel was analyzed by aPhosphorImager, and the amount of cy-cled probe was estimated by integrationof the bands corresponding to intactand cut probe.

RESULTS

Cloning and Enzyme PurificationThe 24-nucleotide junction between

the truncated streptavidin gene and theRNase H gene in pIDB10 was con-firmed by sequencing. The expressedfusion protein contains 293 amino acid(aa) residues. It consists of streptavidinaa residues 16 to 133, followed by thepolylinker (8 aa residues) and the full-length RNase H protein (166 aaresidues) (Figure 2). The fusion proteinwas induced by the addition of M13/T7phage, and the expected polypeptide ofapproximately 32 kDa was obtainedwhen analyzed by SDS-PAGE. The fu-sion protein was purified to apparenthomogeneity with a yield of approxi-mately 1 mg/L of culture.

Structure and Binding Activity ofStreptavidin-RNase H

Thermostability studies of the fu-sion protein at different temperaturesindicated that the molecular mass wasapproximately 128 kDa from 25 to65C and 32 kDa at 100C by SDS-PAGE (Figure 3). These results suggestthat the fusion enzyme is comprised of4 subunits, which are stable at tempera-tures up to at least 65C but are dissoci-ated at 100C. The fusion protein re-tained its biotin binding propertiessince it was purified by an iminobiotinaffinity column. Experiments to test thebinding of the fusion protein to a con-stant amount of probe were performedby incubating the fusion enzyme witheither a biotinylated (ARK2B) or non-biotinylated probe (ARK2) and sub-jecting the products to SDS-PAGE asdescribed in Materials and Methods.


Figure 6. Comparison of the fusion streptavidin-RNase H (Panel A) to the native RNase H (PanelB) in the CPT assay using ARK2B probe and different ratios of probe to enzyme. The autoradi-ogram shows the uncleaved probe (upper band) and the cleaved probe (lower band). Control lanes 1, 5and 9: probe and enzyme without target. Reaction lanes 2, 6 and 10: 0.01 fmol target. Reaction lanes 3, 7and 11: 0.1 fmol target. Reaction lanes 4, 8 and 12: 1 fmol target. C1 is a control with probe but withoutenzyme and target.

Figure 5. Analysis of the thermal stability of native RNase H vs. the streptavidin-RNase H fusion.Enzymes were pretreated at different temperatures (0, 25, 37, 55, 80 and 96C) for 10 min beforesubmitting to the acid-soluble counts assay at 65C.

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Analysis of the products resulting fromthe incubation of ARK2 with the fusionenzyme showed no high molecularweight bands (data not shown). Analy-sis of ARK2B without enzyme alsoshowed no high molecular weightbands (Figure 4, lane 1). Five bandscorresponding to high molecularweight species were observed after in-cubation of ARK2B and the fusion en-zyme (Figure 4, lanes 2, 3 and 4), indi-cating that the biotinylated probe wasbound to the fusion enzyme and was re-tarded during gel migration. The high-est molecular weight species is likelydue to an aggregation of the proteinprobe conjugate. The presence of thefour remaining bands (a, b, c and dfrom higher to lower molecular weight,respectively) are dependent on the ratioprobe:enzyme. Bands a and b are ob-served only with a 1:1 ratio probe:en-zyme, bands c and d with a 1:50 ratioand band d with both 1:50 and 1:5000ratios. These results suggest that with a1:1 ratio, more than two probes arebound to each streptavidin-RNase Hfusion enzyme, and with a 1:5000 ratiothere is one probe bound per fusion en-zyme. Approximately 10% of the probefailed to bind to the enzyme at 1:50 and1:5000 ratios. This may be due to alter-ation or absence of the biotin compo-nent.

RNase H ActivityThe RNase H activity of the fusion

enzyme was compared to the activity ofthe native RNase H using an acid-solu-ble counts assay with M13 DNA/RNAsubstrate. The results showed that thespecific activity of the fusion enzymewas approximately 12% of that of thenative enzyme (data not shown).

A heat-stability study was per-formed to compare the fusion enzymeto the native enzyme. The enzymeswere diluted in glycerol buffer and in-cubated at different temperatures. Fig-ure 5 shows that while the native en-zyme was more stable than the fusionenzyme, the fusion streptavidin-RNaseH was substantially active (approxi-mately 70% of the unheated control)after a 10-min treatment at 65C.

In CPT experiments, the fusionstreptavidin-RNase H was comparedwith the native enzyme using three ra-tios of ARK2B probe:enzyme; 1:1,

1:50 and 1:5000 (Figure 6). At a ratioof 1:1 and 1 fmol of target, strepta-vidin-RNase H was active, whereas thenative RNase H was not. At a ratio of1:50, both enzymes were active with 1fmol of target; however, only the fusionenzyme was active with 0.1 fmol of tar-get. At a ratio of 1:5000 both enzymeswere active. Control reactions (lanes 1,5 and 9) containing enzyme plus probein the absence of target showed noprobe cleavage (Figure 6). This experi-ment shows that the fusion streptavi-din-RNase H was more efficient thanthe native enzyme at low concentra-tions.

The fusion enzyme was further test-ed for activity in CPT experiments us-ing a biotinylated probe (ARK2B) or anon-biotinylated probe (ARK2) (Figure7). At a ratio of 1:1 of probe:enzymeand 1 fmol of target, the fusion enzymewas active with ARK2B but not withARK2. At a ratio of 1:50, the fusion en-zyme was more active with ARK2Bthan ARK2. However at a 1:5000 ratio,the fusion enzyme was more activewith ARK2 than ARK2B. This experi-ment shows that the fusion enzyme atlow concentrations was more efficientwith a biotinylated probe comparedwith a non-biotinylated probe.

DISCUSSION

The streptavidin-RNase H fusionwas shown to be thermostable with anapparent quaternary structure com-posed of 4 subunits. These propertiesare similar to those of the nativestreptavidin (1). The streptavidin-RNase H fusion had both biotin bind-ing and RNase H activities. Similarly, afusion streptavidin-metalloprotein hadbeen produced and was also shown tohave an affinity for both biotin andheavy-metal ions (19). The binding ac-tivity of the fusion streptavidin-RNaseH to a biotinylated oligonucleotide wasshown to be a function of the probe:en-zyme ratio. The streptavidin-RNase Hfusion protein is comprised of four sub-units, each of which can bind one bi-otinylated probe. Our results suggestthat the number of probes bound to thefusion enzyme varied from 1 to 4 andthat the fusion enzyme became saturat-ed with probe at low enzyme concen-trations. The band pattern observed ingel retardation assays is not due to adissociation of the protein, since the fu-sion protein was shown to be one bandwhen stained with silver (data notshown). Furthermore, the quaternarystructure of the streptavidin protein has


Figure 7. Comparison of ARK2 and ARK2B in the CPT assay using the fusion streptavidin-RNaseH and different ratios of probe to enzyme. The percent product represents the amount of cleaved proberelative to the amount of uncleaved probe minus the background observed in the control (probe and en-zyme). These data represent the average of three independent experiments.

been shown to be stable in the presenceof high SDS concentrations (20). Theseresults would indicate that the numberof probes bound to the fusion proteincould be controlled by manipulatingthe ratio of enzyme to probe concentra-tions. For example, RNase H activitymay be affected if four biotinylatedprobes are bound to one streptavidin-RNase H protein. An equilibrium ofone probe per streptavidin-RNase Hprotein might then be preferable.

The fusion enzyme was shown tohave lower activity in an acid-solublecounts assay when compared with thenative enzyme. This decrease in activi-ty may be due to a change in the con-formation of the active site imposed bythe attachment of the streptavidin pro-tein. It may also be due to electrostaticrepulsion of targets caused by closeproximity of the four active sites of afusion protein or steric hindrancecaused by streptavidin subunit associa-

tion. Similar results were previouslyobserved with E. coli RNase H cova-lently attached to an oligonucleotide(11,14). Although the fusion enzymewas less active than the native enzymein an acid-soluble counts assay, the ac-tivities of both enzymes were compara-ble in CPT assays using non-biotinylat-ed probes (data not shown). Thedistinction between the two types of as-says may be explained by differencesin the structure of the substrate and/orthe amounts of the enzyme and sub-strate used in each of the two differentreactions. Indeed, within a molecule ofsubstrate, the acid-soluble counts assayrequires many cleavage events in closeproximity to give a positive activitymeasurment, whereas CPT requiresonly one cleavage event.

With a high ratio of 1:5000probe:enzyme, the fusion was more ac-tive with a non-biotinylated probe com-pared with a biotinylated probe. It is

possible the RNase H enzyme activityis reduced because of attachment of theprobe to the fusion enzyme. However,with a ratio of 1:1 probe:enzyme, theamount of fusion enzyme required foractivity in CPT reactions containing abiotinylated probe was significantlyless than that required for activity witha non-biotinylated probe. Therefore,the streptavidin-RNase H when linkedto the biotinylated probe maintains aclose proximity of the enzyme to thesubstrate and consequently increasesthe efficiency of the reaction.

One disadvantage of the structure ofthe fusion enzyme described is that theenzyme becomes nonfunctional in sub-sequent cycling reactions upon cleav-age of the attached probe. However, ifthe fusion enzyme was attached to abiotinylated target, that enzyme mightparticipate in multiple rounds of probecleavage. Alternatively, the turnoverrate may be increased if the fusion

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enzyme is bound to a biotinylatedoligomer complementary to a sequencethat flanks the target DNA sequence forthe probe. In this configuration the en-zyme will be maintained in close prox-imity to the target and would catalyzethe conversion of multiple probes. Inaddition, this approach would provide asecond order of specificity to the cy-cling reaction since two hybridizationevents (probe and the flanking se-quence with the target DNA strand)must occur to achieve catalytic turn-over of the probe. Experiments are un-der way to examine this approach.

In addition to the increase of the ef-ficiency of CPT, the fusion enzymeshould prove useful as a model forstructural and functional studies ofRNase H and its interaction with sub-strate. RNase H fusion enzyme couldalso be used in antisense studies to pre-vent gene expression. If the fusion en-zyme were bound to an oligonucleotidesequence complementary to the mes-sage, the RNase H enzyme couldcleave the mRNA of interest (15).

ACKNOWLEDGMENTS

We would like to thank T. Atkinson,J. Marostenmaki, D. Schwab and B.Panchuck for oligonucleotide prepara-tion and DNA sequencing. We wouldalso like to thank B. Wu for reviewingthis manuscript and T. Sano and C.Cantor for providing us with the pTSA-18F vector.

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20.Sano, T., M.W. Pandori, C.L. Smith andC.R. Cantor. 1994. Tighter subunit associa-tion of streptavidin upon biotin binding, p. 21-29. In M. Uhlen, E. Hornes and O. Olsvik(Eds.), Advances in Biomagnetic Separation.Eaton Publishing, Natick, MA.

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Received 27 June 1995; accepted 1September 1995.

Address correspondence to:Faouzi BekkaouiID Biomedical Corporation8855 Northbrook CourtBurnaby, BC, V5J 5J1, CanadaInternet: [email protected]


Cycling probe technique

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