Background to the lnvention Genetic disorders are often both progressive and incurable. A pathogenic agent may be expressed from a mutant allele throughout life and its effects are not ameliorated by expression from a normal allele. One therapeutic approach for genetic disorders is to suppress expression from the mutant allele, by targeting the mutant RNA transcripts. However, since the difference between wild-type and mutant gene may be only a single nucleotide, great precision is required in order to target selectively only the mutant transcripts.

The concept of using RNA molecules as therapeutic agents is relatively new. The benefit of using RNA is that it can be designed to be highly specific such that a single nucleotide change within a short RNA molecule can affect the targeting of the RNA molecule.

Small interfering RNAs (siRNAs) are powerful reagents for directed gene expression knockdown (Elbashireta/., Nature, 2001; 411: 494-498). SiRNAs are usually double-stranded RNA molecules, usually 21 nucleotides in length, which target RNA transcripts and block gene expression.

To achieve high levels of gene inhibition, highly effective siRNAs are needed. There is growing evidence to suggest that the internal structure in RNAs has an important role to play in the efficacy of siRNAs, and this is the likely reason why siRNAs targeted to different regions of a gene exhibit different efficacies (Holen et al., Nucleic Acids Res. , 2002; 30: 1757-1766, McManus et<BR> al., J. Immunol., 2002; 169: 5754-5760 and Bahola et al., J. Biol. Chem. , 2003; 278: 15991-15997); inaccessible regions of RNAs are unlikely to make good target sites for siRNAs. Evidence is also emerging that siRNAs can have non- specific side-effects, attributed to sequence similarities with unintended targets (Jackson et al., Nat. Biotech. , 2003; 21: 636-637, Sohail et al., Nuc. Acids. Res.,<BR> 2003; 31: e38 and Saxena etal, J. Biol. Chem. , 2003; 278: 44312-44319), to the<BR> induction of general interferon response (Bridge et a/., Nat. Genet. , 2003; 34: 263-264), or to other cellular proteins (Sledz et al., Nat. Cell. Biol., 2003; 5: 834-

839). Several of these effects are dose-dependent and are exacerbated by higher concentrations of siRNAs. However, finding siRNAs that are effective at low concentrations is difficult and there is a general lack of simple and reliable tools for siRNA discovery.

Therefore, central to the successful application of siRNAs is the ability to <BR> <BR> discover the reagents effective at low concentrations (e. g. , Bridge et al., supra;), and there is a growing need to develop tools for efficient siRNA discovery.

Bahola et al, supra, report the use of a type of DNA array called a scanning array to find accessible sites in mRNAs for designing effective siRNAs.

These arrays comprise oligonucleotides ranging in size from monomers to a certain maximum, usually 20mers, synthesised using a physical barrier (mask) by stepwise addition of each base in the sequence. Thus the arrays represent a full oligonucleotide complement of a region of the target gene. Hybridisation of the target mRNA to these arrays provides an exhaustive accessibility profile of this region of the target mRNA. Such data are useful in the design of antisense oligonucleotides (ranging from 7mers to 25mers), where it is important to achieve a compromise between oligonucleotide length and binding affinity, to <BR> <BR> retain efficacy and target specificity (Sohail et al, Nucleic Acids Res. , 2001 ; 29 (10): 2041-2045).

There is a need for further improvements in methods for identifying suitable siRNA molecules that have high specificity for a target and which can be used effectively at low concentrations to minimise potential side-effects.

Summary of the Invention The present invention is based, in part, on the realisation that effective siRNA molecules can be designed based on an analysis of the extent of hybridisation with a target RNA, and that the extent of hybridisation can be influenced by binding cooperatively acting polynucleotides.

According to a first aspect of the present invention, a method for identifying a polynucleotide molecule capable of binding selectively to a target RNA comprises the steps of : (i) contacting an array of immobilised polynucleotide molecules with a detectably-labelled target RNA; (ii) monitoring the extent to which the target RNA hybridises to the

array; and (iii) identifying the sequence of an immobilised polynucleotide molecule that hybridises to the target, wherein, either prior to or at the same time as step (i), a further polynucleotide is hybridised to the target RNA, and the extent of hybridisation in step (ii) is compared to a control lacking the further polynucleotide.

The extent of hybridisation is measured based on the relative amounts of detectable label. This provides a measure of the accessibility of the RNA to the target and any changes that may have occurred on binding of the further polynucleotide in (iii).

According to a second aspect of the invention, a construct for binding to a RNA target comprises a siRNA molecule having specificity for the target, and a polynucleotide that binds to the target and improves the ability of the siRNA molecule to bind to the target.

According to a third aspect of the present invention, a siRNA molecule comprises a nucleotide sequence corresponding to at least 10 consecutive sequences between positions 308 and 492 of the Epidermal Growth Factor Receptor gene (SEQ ID NO. 1).

According to a fourth aspect of the present invention there is a siRNA molecule as defined above which is used in the manufacture of a medicament for the treatment of a disorder associated with overexpression of the EGFR gene.

According to a fifth aspect of the present invention, a pharmaceutical composition comprises a siRNA molecule and a polynucleotide that acts cooperatively at a target RNA transcript to improve the binding of the siRNA molecule to the target.

Description of the Drawings The invention is described with reference to the accompanying drawings, wherein: Figure 1 (A) shows the hybridisation intensity of various polynucleotides in a dot-blot experiment and (B) is a graphic representation of the hybridisation intensity for each polynucleotide ; Figure 2 (A) shows the hybridisation intensity for various polynucleotides

compared to the intensity exhibited on addition of a cooperatively acting polynucleotide and (B) is a graphic representation of the respective intensities; Figure 3 shows the results of hybridisation intensity for three selected polynucleotides transfected into MDA-MB-231 cells (A) and the resulting expression of the target EGFR gene (B); Figure 4 shows the results of an assay carried out to identify cooperatively acting polynucleotides useful in targeting cyclin B5 mRNA ; and Figure 5 shows the results an experiment to determine the extent of the inhibition of cyclin B5 gene expression using selected polynucleotides.

Description of the Invention The method of the invention is used to identify polynucleotides that act cooperatively with the target RNA to improve binding of the target RNA on the array. Cooperatively acting polynucleotides may therefore be used in conjunction with, for example, a siRNA molecule to target a RNA transcript in vivo. The cooperatively acting polynucleotides act by changing the conformation of the RNA transcript, allowing better access for the siRNA.

Although the invention is described with reference to siRNA molecules, it is envisaged that the methods of the invention can be used to identify/design any polynucleotide molecule that binds to RNA transcripts. This includes polynucleotides that activate RNase H and therefore comprise a segment of DNA as the RNase H substrate.

The present invention makes use of immobilised polynucleotide molecules to form an array that is to be contacted with a target RNA molecule. The formation of polynucleotide arrays is well known in the art, and conventional methods may be used to immobilise the polynucleotides to a suitable support material. For example, the polynucleotides may be modified chemically at either the 3'or 5'end so as to react with a functionalised support surface, forming a covalent attachment. An amine group is a suitable group that may be present at a terminus, and this may be used to react with an aldehyde group on the support material. Alternative procedures for immobilising the polynucleotides will be apparent to the skilled person.

Suitable support materials will also be apparent. For example glass, plastics, ceramics or silicon may be used. The support will usually have a planar

surface and may be any appropriate size. Scanning arrays, as disclosed in Bahola et al., supra, are also suitable.

The polynucleotide immobilised to the support may be of any suitable size.

In a preferred embodiment, the polynucleotide comprises from 10 to 30 nucleotides, more preferably 20 to 25 nucleotides and most preferably 21 to 23, e. g. 21 nucleotides. The design of the polynucleotide immobilised on the support will depend on the particular target under study.

The immobilised polynucleotides may be enzymatically or chemically modified or unmodified DNA or RNA or any other derivations such as locked- nucleic acids. Preferably the polynucleotides are DNA.

The polynucleotides may be produced by known synthesis procedures prior to immobilisation on the support, or may be synthesised on the support. It is preferred that the polynucleotides on the support are of uniform size, although this is not essential.

The sequence of the immobilised polynucleotides will usually correspond to particular sequences on a target gene. For example, for a particular region of a gene under study, a plurality of polynucleotides can be produced, each of the same size and corresponding to different sequences of the gene region. The sequence of the immobilised polynucleotides may overlap or be distinct.

It will be usual to form the arrays with all of the different complements of the selected target sequence, given the size of arrayed molecule. The array will usually comprise distinct clusters of polynucleotides of the same type, thereby providing many hybridisation targets for the target sequence and allowing the extent of hybridisation to be more readily determined. High density polynucleotide arrays are well known in the prior art.

The target RNA is provided in a form that is"detectably-labelled", i. e. the RNA incorporates a moiety that permits detection. Radiolabels are preferred as they permit simple detection and can be incorporated into the RNA molecule without affecting the RNA structure. A preferred radiolabel is 33P. Fluorescent molecules may also be used, as will be appreciated by the skilled person.

According to the invention, one or more target RNA molecules are contacted with the arrayed immobilised polynucleotides, under conditions that permit hybridisation between complements.

Either prior to, or at the same time as the step of introducing the target RNA, one or more further polynucleotides are brought into contact with the target RNA.

The arrayed immobilised polynucleotides will usually be designed to represent potential RNA inhibitor molecules and the sequences of suitable arrayed polynucleotides will be used in the design of, for example, siRNA molecules. In this embodiment, the further polynucleotide intended for <BR> <BR> hybridisation to the target (i. e. that referred to in step (iii) ) is a cooperatively acting polynucleotide. Preferably this cooperatively acting polynucleotide is present in excess of the target RNA.

However, in a separate embodiment, the immobilised polynucleotides may represent different potential cooperatively-acting polynucleotides, and therefore the method of the invention is used to identify sequences that act cooperatively.

In this embodiment, RNA inhibitor molecules may be hybridised to the target either prior to or (preferably) at the same time as contacting the array with the target RNA. The RNA inhibitor molecules may be the same or different.

Polynucleotides that act cooperatively on the target RNA to improve binding to the array can be identified, based on a comparison with a control reaction carried out in the absence of the putative cooperatively acting polynucleotide. Suitable cooperatively acting polynucleotides can then be used in therapy in conjunction with RNA inhibitor molecules.

Cooperatively-acting polynucleotides can be designed with any suitable sequence that hybridises in proximity to the target site for the anti-sense inhibitor molecule. Accordingly, in the design of cooperatively-acting polynucleotides, an understanding of the target sequence will be important, and different polynucleotides can be designed to bind to various regions on the target, to identify those that exhibit a cooperative effect.

The cooperatively acting polynucleotides may be detectably labelled to differentiate between polynucleotides of different sequence during the method of the invention.

Typically hybridisation is carried out at a temperature in the range 20°C- 37°C. Hybridisation at 37°C is preferred as this is the in vivo temperature at which the siRNAs are intended to function. Conventional hybridisation reagents

may be used, as will be appreciated by the skilled person.

Having identified a suitable polynucleotide sequence that binds to the target RNA in conjunction with the cooperatively acting polynucleotide, it will be readily apparent to the skilled person how to produce RNA inhibitors, e. g. siRNA molecules, based on the determined polynucleotide sequence. As stated previously, the present invention can be used to design any suitable RNA inhibitor, including siRNA molecules. A review of RNA inhibitor molecules is contained in Uhimann et al., Chemical Reviews, 1990; 90 (4): 544-584. SiRNA molecules are preferred. SiRNA molecules may be synthesised using chemical or enzymatic techniques. The siRNA will usually be used as a double-stranded molecule, and so the complementary sequence can be synthesised in the same manner.

There are now many companies that provide the necessary reagents to synthesise RNA molecules or provide a contract service forthe custom synthesis of siRNA molecules.

Stability of the siRNA or the cooperatively acting polynucleotide may be improved by known techniques to protect against nuclease cleavage when administered to a patient. For example, the polynucleotides may be designed with modified internucleotide linkages that are resistantto nuclease degradation, e. g. phosphorothioate linkages. Separately, or in addition, other modifying groups may be incorporated, including 2'-0-alkyl groups on the pentose sugar, to improve hybridisation with the target and to protect against endo and exonuclease attack. This will all be appreciated by the skilled person.

The siRNA molecules and/orthe cooperatively acting polynucleotides may be formulated into any suitable pharmaceutical composition. Suitable compositions are known, based on conventional siRNA formulations.

The siRNAs (and cooperatively acting polynucleotides) can be administered to a patient using any convenient route of administration, including intravenous, intramuscular and subcutaneous administration. Administration to a patient will be in any therapeutically-effective amount.

SiRNA molecules (and cooperatively acting polynucleotides) may be formulated with reagents which promote the transfection of the molecules into the cell nucleus. Suitable reagents will be apparent to those skilled in the art.

The siRNA (and/or cooperatively acting polynucleotides) may also be incorporated into cloning vectors, plasmids, viral or other such constructs, which can be inserted into a cell and used to produce the siRNA molecules and cooperatively acting polynucleotides (such as in the form of short RNA fragments). Suitable vectors are described in Tuschl, Nature Biotechnology, 2002; 20 (5): 446-448, Brummelkamp et a/., Science, 2002; 296: 550-553, and Paul et al., Nature Biotechnology, 2002; 20: 505-508.

The methods of the invention are further described in the following Examples. The Examples make use of arrays fabricated using the known sequence for the epidermal growth factor receptor (EGFR).

Example 1 Array fabrication: Twenty 21-mer DNA polynucleotides and twenty corresponding 19-mer DNA polynucleotides, (each shortened by two nucleotides at the 3'end of the 21-mers) from the first-0. 55kb nucleotides sequence of the coding region of the epidermal growth factor receptor (EGFR) mRNA were designed (Table 1). The polynucleotides were modified to contain a 3'amine group for terminal attachment (Qiagen Ltd). 500nl of a 100, uM oligonucleotide solution in 3xSSC was spotted onto aldehyde-coated glass slides (Genetix). The slides were incubated in a humid chamber for-5hrs and, thereafter, were processed largely according to the supplier's information.

Table 1: 21-mer polynucleotides OLIGO Sequence 5'--> 3' EG1 Tac tcg tgc ctt ggc aaa ctt-C6NH (SEQ ID NO. 2) EG2 Tga gct tgt tac tcg tgc ctt-C6NH (SEQ ID NO. 3) EG3 Agt gcc caa ctg cgt gag ctt-C6NH (SEQ ID NO. 4) EG4 Gga ggc tga gaa aat gat ctt-C6NH (SEQ ID NO. 5) EG5 Aag gac cac ctc aca gtt att-C6NH (SEQ ID NO. 6) EG6 Cac ata ggt aat ttc caa att-C6NH (SEQ ID NO. 7) EG7 Taa gaa gga aag atc ata att-C6NH (SEQ ID NO. 8) EG8 Agc cac ctc ctg gat ggt ctt-C6NH (SEQ ID NO. 9) EG9 Tga ggg caa tga gga cat aac-C6NH (SEQ ID NO. 10)

EG10 Agg aat tcg ctc cac tgt gtt-C6NH (SEQ ID NO. 11 EG11 Tcc tct gat gat ctg cag gtt-C6NH (SEQ ID NO. 12) EG12 Gga att ttc gta gta cat att-C6NH (SEQ ID NO. 13) EG13 Tag ata aga ctg cta agg cat-C6NH (SEQ ID NO. 14) EG14 Ggc agc tcc ttc agt ccg gtt-C6NH (SEQ ID NO. 15) EG 15 Att tct cat ggg cag ctc ctt-C6NH (SEQ ID NO. 16) EG16 Cgc cat gca gga ttt cct gta-C6NH (SEQ ID NO. 17) EG17 Ggg ttg ttg ctg aac cgc acg-C6NH (SEQ ID NO. 18) EG18 Cca ctg gat gct ctc cac gtt-C6NH (SEQ ID NO. 19) EG19 Aaa gtc act gct gac tat gtc-C6NH (SEQ ID NO. 20) EG20 Aag tcc atc gac atg ttg ctg-C6NH (SEQ ID NO. 21) Hybridisation and image analysis : Messenger RNA (mRNA) transcripts were prepared by in vitro transcription. A PCR product corresponding to the 3.6 kb full-length EGFR mRNA containing a promoter sequence for T7 RNA polymerase was used as a template. In vitro transcriptions were carried out in the presence of a-33P UTP for internal labelling of the transcripts. Hybridisations were performed in 1 M NaCI, 1 OmM Tris pH 7.4, 1 mM EDTA, 0.01 % SDS w/v, for 3 hours at 37°C.

To test for polynucleotides that may produce conformational changes in the target RNA structure ('cooperative polynucleotides"), polynucleotides were mixed with the target RNA in large excess (-100 : 1 ratio) and were allowed to hybridise with target RNA in solution for 15-20 minutes. The polynucleotide- transcript complex was then hybridized with the array.

The cooperative polynucleotide used in this experiment had a sequence corresponding to SEQ ID NO. 12 (EG11).

A control experiment was also carried out, in the absence of the cooperative polynucleotide.

The slides were washed in the hybridisation buffer for 5-10 seconds, dried in layers of filter paper and exposed to a phosphor storage screen overnight.

The screen was scanned on a STORM Phosphorlmager. The hybridisation intensities of the spots were quantified using ImageQuant.

The image analysis of the slides identified spots which had a more intense signal when the cooperative polynucleotide was present compared to the control spot where no cooperative polynucleotide had been used. This demonstrated that the polynucleotide had a cooperative effect on certain target polynucleotides.

The experiments identified a certain region on the EGFR as being of particular utility as a target for siRNA therapies. The sequence resides between positions 308 and 492 of the EGFR gene sequence (SEQ ID NO. 1).

SiRNA molecules that bind within this region are useful to treat disorders associated with the activation of EGFR signaling, including, among others, cell proliferative disorders and angiogenesis.

Of the arrayed polynucleotides identified in Table 1, those identified as EG2, EG12, EG14, EG15, EG17 and EG18 showed the most hybridisation to the target RNA (as shown in Figure 1) and made the most suitable sequences forthe design of a siRNA molecule.

Figure 2 shows the results obtained when using the cooperative polynucleotide (referred to in Figure 2 as a facilitator). The cooperative polynucleotide increased the effectiveness both the 21-mer and 19-merversions of EG12 (SEQ ID NO. 13), resulting in improved hybridisation.

Validation of data in cell culture A cell-based assay system was used to test the effectiveness of siRNAs polynucleotides derived from the hybridisation assessment of polynucleotides in the arrays. Chemically synthesised siRNAs corresponding to sequences EG2, EG14 and G17, were obtained from commercial suppliers (Dharmacon). Each siRNA had a 19 base pair duplex and 2 nt 3'end overhangs. The antisense sequence of each siRNA was identical to its corresponding polynucleotide sequence in the array, except that siRNAs were made of ribonucleotide monomer, instead of deoxyribonucleotide monomer of DNA polynucleotides.

Table 2; List of siRNAs siRNA 2 Sense 5'GGCACGAGUAACAAGCUCATT- (SEQ ID NO. 26) Antisense 3'TTCCGUGCUCAUUGUUCGAGU- (SEQ ID NO. 27) siRNA 14 Sense 5'CCGGACUGAAGGAGCUGCCTT- (SEQ ID NO. 28) Antisense 3'TTGGCCUGACUUCCUCGACGG- (SEQ ID NO. 29)

siRNA 17 Sense, 5'UGCGGUUCAGCAACAACCCTT- (SEQ ID NO. 30) Antisense 3'GCACGCCAAGUCGUUGUUGGG- (SEQ ID NO. 31) The siRNAs were transfected into MDA-MB-231 cells and were tested for their ability to downregulated the expression of EGFR in the cells by western blotting as described below.

MDA-MB-231 cells were grown in RPMI-1640 medium (InVitrogen) supplemented with 10% heat-inactivated FCS (Fetal Calf Serum), 2mM L- Glutamine, 40ug/ml Streptomycin and 40U/ml Penicillin (Sigma). Cells were grown at 37°C in 5% CO2. siRNAs corresponding to EG2, EG14 and EG17 were transfected into MDA-MB-231 cells at 50nM concentration (unless otherwise mentioned) using Oligofectamine (InVitrogen). 5x106cells/6cm dish were seeded in 5ml medium to achieve the optimal confluency (30-40%) fortransfection on the following day. The mixes were prepared as below ; Tube 1: x ul of siRNA stock (to obtain 50nM final concentration) + (560. 5ui-x u !) of Optimem (InVitrogen) Tube 2: 4. 5pl Oligofectamine + 150u1 Optimem The two tubes were incubated at room temperature for 10 minutes, mixed and incubated for a further 25 minutes to obtain a siRNA-Oligofectamine complex. During the incubation cells in 6cm plates were washed with 4m ! of PBS (ICN). Then 285pl of Optimem and 715u1 siRNA-Oligofectamine complex were added to each plate. The cells were incubated for 4 hours. 100, u1 of FCS and 4ml of complete medium were added to each dish and the cells were incubated at 37°C in 5% C02 until they were lysed.

Preparation of cell lysates and protein estimation The cells were washed with 5mi of ice-cold PBS. 1 mi of ice-cold PBS was then added to the dishes, the cells were scraped and collected in 1. 5mi microcentrifuge tubes and centrifuged for 30 seconds at 13,000 rpm at 4°C. The pellet was re-suspended in 30-150psi of lysis buffer depending on the pellet size

and left on ice to lyse for 30-60 minutes. Cell debris was removed by centrifugation at 13,000 rpm at 4°C for 20 minutes.

For protein estimation, a Bio-Rad Protein Assay Kit-il was used as instructed by the manufacturer. BSA (Bovine Serum Albumin) was used as a standard protein. The volume of lysate, which contained 30mg/ml of standard protein, was calculated and loaded into each well for electrophoresis.

Western blotting To determine EGFR protein expression levels SDS-PAGE separation of samples was performed, followed by Western blotting. Briefly, protein samples were heated to 95° for 5 minutes, and then 30ug protein sample in sample buffer (InVitrogen) was loaded into preformed wells along with a pre-stained high range molecular weight marker (InVitrogen). Samples were separated in a 10% NuPAGE Bis-Tris gel (InVitrogen), in SureLock Mini-Cell apparatus (InVitrogen) for 1 hour 30 minutes at 200V prior to being transferred to nitrocellulose Hybond- C membrane (Amersham Pharmacia Biosciences) at 30V for 2 hours.

Transferred proteins were blocked with 5% non-fat dried milk in PBS-Tween (0. 1 % v/v) and subsequently probed for EGFR using primary anti-human EGFR antibody raised in rabbit (Cell Signalling Technologies) for 4 hours at room temperature. The primary antibody was further probed with Peroxidase- Conjugated Goat Anti-Rabbit Immunoglobulins (DAKO) which was then detected using ECL Plus reagents (Amersham Pharmacia Biosciences) and autoradiography (Kodak film) according to the manufacturer's instructions. To ensure equal loading of proteins P-tubulin levels were also detected using primary Monoclonal Anti-ß-tubulin antibody (Mouse Antibody) (Sigma) and the secondary Peroxidase-Conjugated Goat Anti-Mouse Immunoglobulins (DAKO).

The band intensities were quantified using AlphaEaseFC 3.1. 2 (Alpha Innotech).

The results in Figure 3 show that the siRNA corresponding to the polynucleotide producing the strongest signal in the set was the most effective in inhibiting the expression of EGFR. This suggests that such polynucleotide arrays could be used to design effective siRNAs.

Example 2 RNase H/dN, 2assay An RNase H/polynucleotide library (dN12) assay (Sohail et a/., Adv.

Biochem. Eng. Biotechnol. 2002; 77: 43-56) was used to find polynucleotides which, on binding, may produce structural changes in mRNA (cooperative polynucleotides). First, radio-labelled cyclin B5 mRNAtranscriptofXenopuswas hybridised to a scanning array of antisense polynucleotides (Sohail et a/., 2001 supra) to determine the binding efficiencies of the various polynucleotides. The scanning array was complementary to the first 111 nt sequence of the coding region of cyclin B5 mRNA, comprising polynucleotides ranging in size from monomers to a maximum of 21-mers (Figure 4; SEQ ID NO. 22). The hybridisation revealed regions with various levels of accessibility. A 16-mer polynucleotide (F2; SEQ ID NO. 23) was selected from a region that showed moderate level of hybridisation and its 2'0 methyl modified analogue (F20ME) used in an RNase H/dN12 cleavage assay.

F20ME was hybridised with a 32P-end labelled in vitro transcript of cyclin B5 and this mix was used in an RNase H cleavage/dN, 2assay.

Analysis of the reaction by polyacrylamide gel electrophoresis revealed a site in B5 RNA (N: Figure 4) within the first 100 or so nucleotides showing enhanced cleavage by RNase H in the presence of F20ME, but not in its absence (Figure 4). The exact location and sequence of this site was determined by using the scanning array of anti-B5 polynucleotides as follows.

Split hybridisation to the anti-B5 polynucleotide scanning array Polynucleotide scanning arrays are symmetrical above and below the centreline (Sohail et al., 2001 supra). Therefore, each half of the array can be hybridised with a different solution simply by splitting with a rubber tube placed atthe centreline (Figure 4). Thus the hybridisation behaviouroftwo nucleic acids with a set of polynucleotides can be studied in parallel under similar experimental conditions.

Cyclin B5 mRNA transcript was hybridised to one half of the array, and to the other was hybridised a mixture of B5 transcript and F2. Image analysis revealed considerable differences in the hybridisation of the two mRNA transcripts. The presence of F2 considerably enhanced accessibility of a neighbouring region of RNA, supporting the similar observation in the RNase H assay. Using the computer program, xvseq, it was possible to identify the precise sequences of the polynucleotides in the array that exhibited a change in

the hybridisation. As expected, with the addition of F2, the hybridisation of mRNA at the site corresponding to this sequence in the array dropped to almost background level, since the polynucleotide in solution occupied most of the sites in the hybridising mRNA transcript (Figures 4B & C).

Validation of hybridisation data in a cell lysate assay A cell-free system was then used to test whether the changes in the accessibility of a region of RNA in the presence of F2, as observed in the RNase assay and by hybridisation to the polynucleotide array, could also be reproduced under near-physiological conditions. This was achieved by performing an RNase H-mediated cleavage assay in HeLa cell lysate supplemented with 1 x RNase H buffer.

Two antisense polynucleotides, N2 (SEQ ID NO. 24) and N6 (SEQ ID NO.

25), complementary to the region of RNA that exhibited a change in accessibility were selected (Figure 4) and their ability to cleave B5 RNA in the presence or absence of F20ME was investigated. 2'0 methyl modification of F2 allowed changes in cleavage efficiencies to be observed with NO, N2 and N6 without cleaving the RNA at F2 binding site. 32P-End-labelled cyclin B5 mRNA transcript was mixed with F20ME plus either NO, N2 or N6 polynucleotide and was added to the cell-free reaction mix. After incubation for 1 hour, the reactions were treated with Proteinase K and extracted twice with phenol/chloroform to remove proteins and were analysed in a 7% denaturing polyacrylamide gel. The results of this assay are shown in Figure 5. In the absence of F20ME, NO and N2 showed similar efficacies and N6 showed higher efficacy than NO and N2 as expected from the array hybridisation data. RNase H-mediated cleavage at N2 and N6 binding sites was considerably increased in the presence of F20ME. As expected cleavage at the NO site was not affected by the presence of F20ME.

These data suggest that cis-binding effector polynucleotides can be used to enhance cleavage by an anti-mRNA reagents at a neighbouring binding site and that scanning arrays are useful tools to discover such cooperativelyworking pairs of anti-mRNA reagents.