WO2005024061A2 - Compounds and methods for modulation of dna replication - Google Patents

Abstract

Molecules and methods for modulating chromosomal DNA replication and cell proliferation and assays to identify agents for modulating chromosomal DNA replication and cell proliferation. The molecules, methods and assays may be used in the treatment of proliferative diseases such as cancer.

Description

COMPOUNDS AND METHODS FOR MODULATION OF DNA REPLICATION

The present invention relates to molecules and methods for modulating chromosomal DNA replication and cell proliferation and assays to identify agents for modulating chromosomal DNA replication and cell proliferation. More specifically the molecules, methods and assays of the present invention may be used in the diagnosis and treatment of proliferative diseases such as cancer.

INTRODUCTION

The accurate and timely duplication of the genome is a major task for eukaryotic cells. This process requires the cooperation of multiple factors to ensure the stability of the genetic information of each cell. Mutations, rearrangements, or loss of chromosomes can be detrimental to a single cell as well as to the whole organism, causing failures, disease, or death. On the other hand control of DNA replication itself plays a major part in the development of diseases such as cancer. For these reasons regulation and mechanisms of the initiation of eukaryotic DNA replication have been intensively investigated. Over the years numerous studies were carried out to find the essential factors involved in the process and to determine their functions during DNA replication. These studies gave rise to a model of the organization and the coordination of DNA replication within the eukaryotic cell.

The initiation of DNA replication at the G1 to S phase transition is a key regulatory step of the cell division cycle in eukaryotic cells. Once DNA replication has initiated, control mechanisms ensure that the entire genomic DNA is replicated precisely once, and after completion, one replicated genome segregates to each of the two daughter cells during mitosis (for reviews, see Refs. 52-57). Cell fusion experiments in mammalian somatic cells established that G1 , but not G2 phase nuclei, initiate DNA replication prematurely when exposed to an S phase cytosolic environment (58). Key regulators of initiation were identified in genetic and cytological experiments in vivo. Roles for cyclin- dependent protein kinases (Cdks) and their G1 and S phase-specific regulatory subunits cyclin D, E, and A in inducing DNA replication have been documented (59-68). The analysis of initiation of DNA replication in vivo was recently complemented by a biochemical approach through the establishment of cell-free systems from human and mammalian cells (28, 31 , 29). DNA replication is initiated in nuclei isolated from human G1 , but not G2 phase cells when incubated in S phase cytosolic extract and S phase-specific nuclear factors. A nuclear extract could be substituted by purified recombinant cyclins A and E, complexed to their kinase partner Cdk2, to initiate DNA replication, directly demonstrating functional roles for these nuclear cyclin/Cdks complexes (28). These cyclin/Cdks were essential, but not sufficient, as nuclei also required soluble factors present in a cytosolic extract from S phase cells to initiate replication (28).

Late G1 phase nuclei from cells synchronized by release from either mitosis or quiescence are relatively undefined heterogeneous and dynamic populations as a result of cells passing the state of competence at the time of preparation (28, 31). A significant step toward molecular and temporal dissection of the establishment of DNA replication forks in human somatic cells is the availability of defined populations of homogeneous template nuclei reversibly arrested in a state of replication initiation competence. More specifically, template nuclei were used which were isolated from cells synchronised in late G1 phase by a treatment with the plant compound mimosine (34). It was shown before that these nuclei initiate semi- conservative DNA replication very efficiently in human cell extracts in an origin-dependent manner (29,30).

Initiation of chromosomal DNA replication in late G1 phase nuclei depends on incubation in a cytosolic extract from proliferating cells (29). In the absence of this extract, or the factors contained in it, no new DNA replication foci are initiated in vitro but DNA replication can proceed further at replication forks that were established in vivo and that are clustered into foci prior to the isolation of the nuclei (28-30,34). By fractionating the initiating cytosolic extract, RPA was identified as one of the essential factors required to trigger initiation of new DNA replication foci in late G1 phase nuclei (95). This inductive functional assay provides strong direct evidence that soluble RPA originally present in the cytosolic extract is required to trigger replication in vitro and is recruited to forming replication foci within the nuclear templates. These observations have added the context of chromosomal DNA replication in nuclei of human somatic cells to the list of systems that require RPA as a replication initiation factor. Apart from the strict requirement to initiate DNA replication, the precise molecular mechanism of RPA involvement in the initiation of chromosomal DNA replication is not yet defined.

Furthermore, from these experiments it is clear that other factors are required to trigger initiation of DNA replication. However, up to now there is no suggestion regarding the nature or number of such factors.

DESCRIPTION OF THE INVENTION

In general, the present invention is based on the novel finding that a specific class of small RNAs is involved in the initiation of DNA replication, and that inhibition of said small RNA function results in inhibition of DNA replication.

As discussed in more detail below we have surprisingly identified initiation factors, which were previously not implicated in the regulation of mammalian chromosomal DNA replication. Even more surprisingly, it turned out that these initiation factors were small RNAs and not proteins as generally assumed. In particular we show here that the small RNAs U2 snRNA, 5S rRNA, hY1 RNA, hY3 RNA, hY4 RNA and hY5 RNA, in particular hY1 RNA, hY3 RNA, hY4 RNA and hY5 RNA are involved in the initiation of chromosomal DNA replication in human cells. This is unprecedented because no model system so far has identified a role for a discrete RNA species in eukaryotic chromosomal DNA replication, and thus our predicted results will allow to refine our understanding of the regulation of chromosome replication and hence the maintenance of genetic integrity during cell proliferation in somatic human cells.

U2 snRNA is a small nuclear RNA known from the splicosome which is responsible for the removal of introns from the pre-mRNA (gene see figure 7, RNA sequence see figure 8D) (93). 5S is known to be part of the large subunit of the eukaryotic ribosome (gene sequence see figure 6, RNA sequence see figure 8C) (94). HY1 RNA (gene sequence see figure 17, RNA sequence see figure 19A), hY3 RNA (gene sequence see figure 18, RNA sequence see figure 19B), hY4 RNA (gene sequence see figure 4, RNA sequence see figure 8A) and hY5 RNA (gene sequence see figure 4, RNA sequence see figure 8B) are found free or as part of the Ro ribonucleoprotein particles (Ro RNPs). In particular, these Ro RNP particles are known because of their antigenecity for autoimmune antibodies in patients suffering form systemic lupus erythematosis.

As indicated above, none of these RNAs, or the proteins that are known to be associated with them, have been previously implicated in DNA replication. In fact, no functional role for any Y RNA has been described in the literature to date.

Of particular interest is the newly discovered functional role of these RNAs in view of various applications in the diagnosis and treatment of proliferative diseases, in particular cancer. It will be appreciated from the foregoing that U2 snRNA, 5S rRNA, hY1 RNA, hY3 RNA, hY4 RNA and hY5 RNA are drug targets in the treatment of proliferative diseases, in particular cancer. In particular, every agent, which would inhibit the full function of one or more of these RNAs could be useful in the treatment of such a disease. This inhibition could be for example, conveyed by inhibiting the binding with other components of the DNA replication initiation complex, or by destruction of the said RNAs. Therefore, it is an object of the present invention to make use of these unexpected findings in the field of diagnosis and therapy for proliferative diseases.

The first aspect of the present invention is an agent that modulates chromosomal DNA replication by modulating the function of hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and/or hY5 RNA, preferably hY1 RNA, hY3 RNA, hY4 RNA and/or hY5 RNA. Preferably, the agent of the present invention inhibits chromosomal DNA replication. In another aspect the agent of the present invention increases chromosomal DNA replication.

The "function of hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and hY5 RNA" is herein understood as the effect of these RNAs on DNA replication. Therefore an agent which decreases DNA replication is an agent which prevents the effect of hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and/or hY5 RNA, respectively, on initiation of DNA replication directly, for example by degradation of said RNAs, chemical modification of said RNAs or inhibition of expression of said RNAs or indirectly, for example by inhibiting the binding with other initiation factors. On the other hand, an agent which increases the function of hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and/or hY5 RNA is an agent which increases the effect of hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and/or hY5 RNA, for example by mimicking hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and/or hY5 RNA function or by increasing the half-life of said RNAs or by stabilising the interaction of said RNAs with other initiation factors.

An agent according to the present invention can be designed or screened or identified by its ability to mimic hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA function. This includes analogues of hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and hY5 RNA, which are modelled to resemble the three dimensional structure of hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and hY5 RNA. These analogues could be either designed in a way to inhibit hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and hY5 RNA function by competing with said endogenous RNAs or to increase hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and hY5 RNA function by mimicking these molecules.

In further aspect an agent according to the present invention is an antibody, preferably an antibody against hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA comprising complexes.

The term "antibody" as used herein includes but is not limited to polyclonal, monoclonal, chimaeric, single chain, Fab fragments and fragments produced by a Fab expression library. Such fragments include fragments of whole antibodies which retain their binding activity for a target substance, Fv, F(ab') and F(ab')2 fragments, as well as single chain antibodies (scFv), fusion proteins and other synthetic proteins which comprise the antigen-binding site of the antibody. Furthermore, the antibodies and fragments thereof may be humanised antibodies, for example as described in US-A-239400.

Neutralising antibodies, i.e., those which inhibit biological activity of the substance polypeptides, are especially preferred for diagnostics and therapeutics.

Antibodies may be produced by standard techniques, for example by immunisation with the appropriate fragment of hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA comprising complexes, or by using a phage display library.

If polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, horse, etc) is immunised with an immunogenic polypeptide bearing a epitope such as the particular hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA comprising complexes described herein.

Monoclonal antibodies directed against particular epitopes, such as the particular domains or fragments of hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA comprising complexes described herein, can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies by hybridomas is well known. Immortal antibody-producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. Panels of monoclonal antibodies produced against orbit epitopes can be screened for various properties; i.e., for isotype and epitope affinity.

Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents (84, 85).

Antibody fragments which contain specific binding sites for the substance may also be generated. For example, such fragments include, but are not limited to, the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulphide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (86).

An agent according to the present invention may be an agent, which modulates expressions of one or more genes selected from the group of hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and hY5 RNA.

Preferably, the invention includes an agent, which inhibits hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA gene expression. Alternatively, the invention includes an agent, which stimulates hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA gene expression.

In a preferred aspect the agent comprises an antisense RNA, a small interfering RNA, or an engineered transcription repressor that inhibits chromosomal DNA replication gene transcription.

A preferred object of the invention is therefore an oligonucleotide, which is at least partly complementary to hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA or their genes. For example, preferred oligonucleotides according to the present invention are depicted in figures 9-16, 20-25.

Agents that inhibit hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA gene expression include antisense RNA, small interfering RNAs and ribozyme molecules which selectively cleave polynucleotides encoding hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA (87, 88, 89).

Agents that inhibit or stimulate hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA gene transcription can be designed, for example using an engineered transcription repressor, or they can be selected, for example using the screening methods described in later aspects of the invention (90, 91).

RNA interference (RNAi) is the process of sequence-specific post- transcriptional gene silencing in animals initiated by double-stranded (dsRNA) that is homologous in sequence to the silenced gene. The mediators of sequence-specific mRNA degradation are typically 21- and 22-nucleotide small interfering RNAs (siRNAs) which, in vivo, may be generated by ribonuclease III cleavage from longer dsRNAs. It was shown that 21- nucleotide siRNA duplexes specifically suppress expression of both endogenous and heterologous genes in, for example, mammalian cells (92). In mammalian cells it is believed that the siRNA has to be comprised of two complementary 21mers as described below since longer double-stranded (ds) RNAs will activate PKR (dsRNA-dependent protein kinase) and inhibit overall protein synthesis.

Duplex siRNA molecules selective for hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA can readily be designed by reference to the hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA cDNA sequence. For example, they can be designed by reference to the human hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA cDNA, or naturally occurring variants thereof. Once designed, the synthetic RNA molecules can be synthesised using methods well known in the art.

siRNAs may be introduced into cells in the patient using any suitable method. Typically, the RNA is protected from the extracellular environment, for example by being contained within a suitable carrier or vehicle. Liposome- mediated transfer is preferred. Liposomes are described in more detail with respect to antisense nucleic acids below. It is particularly preferred if the oligofectamine method is used.

Antisense nucleic acid molecules selective for hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA can be designed by reference to the cDNA or gene sequence, as is known in the art.

Antisense nucleic acids, such as oligonucleotides, are single-stranded nucleic acids, which can specifically bind to a complementary nucleic acid sequence. By binding to the appropriate target sequence, an RNA-RNA, a DNA-DNA, or RNA-DNA duplex is formed. These nucleic acids are often termed "antisense" because they are complementary to the sense or coding strand of the gene. Recently, formation of a triple helix has proven possible where the oligonucleotide is bound to a DNA duplex. It was found that oligonucleotides could recognise sequences in the major groove of the DNA double helix. A triple helix was formed thereby. This suggests that it is possible to synthesise sequence-specific molecules, which specifically bind double-stranded DNA via recognition of major groove hydrogen binding sites.

By binding to the target nucleic acid, the above oligonucleotides can inhibit the function of the target nucleic acid. This could, for example, be a result of blocking the transcription, processing, poly(A)addition, replication, translation, or promoting inhibitory mechanisms of the cells, such as promoting RNA degradations.

Typically, antisense oligonucleotides are 15 to 35 bases in length. However, it is appreciated that it may be desirable to use oligonucleotides with lengths outside this range, for example 10, 11 , 12, 13, or 14 bases, or 36, 37, 38, 39 or 40 bases.

Antisense oligonucleotides may be administered systemically. Alternatively the inherent binding specificity of oligonucleotides characteristic of base pairing is enhanced by limiting the availability of the oligonucleotide to its intended locus in vivo, permitting lower dosages to be used and minimising systemic effects. Thus, oligonucleotides may be applied locally to achieve the desired effect. The concentration of the oligonucleotides at the desired locus is much higher than if the oligonucleotides were administered systemically, and the therapeutic effect can be achieved using a significantly lower total amount. The local high concentration of oligonucleotides enhances penetration of the targeted cells and effectively blocks translation of the target nucleic acid sequences.

It will be appreciated that antisense agents also include larger molecules which bind to hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA or their genes and substantially prevent expression of hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA. Thus, an antisense molecule which is substantially complementary to hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA is envisaged as part of the invention. The larger molecules may be expressed from any suitable genetic construct and delivered to the patient. Typically, the genetic construct which expresses the antisense molecule comprises at least a portion of the hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA or their gene(s) operatively linked to a promoter which can express the antisense molecule in the cell.

Although genetic constructs for delivery of oligonucleotides can be DNA or RNA it is preferred if it is DNA. Equivalent genetic constructs can be used to deliver antisense oligonucleotides to a patient as described above in relation to the delivery of oligonucleotides encoding hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA.

Preferably, the genetic construct is adapted for delivery to a human cell. In a preferred aspect, the oligonucleotide which is antisense further comprises a vector which is designed to express antisense RNA. Hence, the invention further provides a oligonucleotide comprising a nucleic acid sequence which is antisense to a oligonucleotide encoding the hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA for use in medicine, especially in the manufacture of a medicament for treating cancer.

Ribozymes are RNA or RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity. For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate. This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.

Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage/ligation reactions involving nucleic acids. For example, US Patent No 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes. Thus, sequence-specific ribozyme-mediated inhibition of gene expression may be particularly suited to therapeutic applications, and may be designed by reference to the DNA, which is a copy of the RNA to be cleaved (e.g. the human hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA, or naturally occurring variants thereof).

The invention also includes variants of the oligonucleotides encoding the agents described above, or variants of the antisense oligonucleotides, or variants of the siRNAs.

The term "variant" includes oligonucleotides having at least 90%, preferably at least 91%, or at least 92%, or more preferably at least 93%, or at least 94%, or at least 95%, or at least 96%, or yet more preferably at least 97%, or at least 98%, or most preferably at least 99% sequence identity with the oligonucleotides encoding the agents described above, or the antisense oligonucleotides, or the siRNAs.

The term "variant" also encompasses sequences that are complementary to sequences that are capable of hybridising under highly stringent conditions (eg 65°C and O.lxSSC {IxSSC = 0.15 M NaCI, 0.015 M Na3citrate pH 7.0}) to oligonucleotides encoding the agents described above, or to the oligonucleotide agents.

A preferred aspect of the present invention is an agent, which is an RNA comprising a double stranded structure, which is substantially identical to at least part of a target gene selected from the group of hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and hY5 RNA. Preferred objects of the invention are shown in figures 9-12, 20-21.

A further aspect of the present invention is a DNA template encoding the nucleic acids of the present invention, preferably a vector comprising the DNA template encoding a nucleic acid of the present invention. As it will be appreciated by the skilled person a preferred vector is a delivery vector which is specific for tumour cells.

A further aspect of the present invention provides a pharmaceutical composition comprising an agent which inhibits a hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA activity and a pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof). The invention includes a pharmaceutical composition comprising a oligonucleotide that encodes an agent which modulates hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA activity and a pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof).

The pharmaceutical compositions may be for human or veterinary medicine and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier, or excipient. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder, lubricant, suspending agent, coating agent, solubilising agent.

A further aspect of the present invention is a pharmaceutical preparation comprising an agent, a DNA template or a vector of the present invention.

A further aspect of the present invention is the use of hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA in the diagnosis of a proliferative disease.

A further aspect of the present invention is the use of an inhibitor of hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA mediated initiation of chromosomal DNA replication for the preparation of a medicament for treating a proliferative disease.

A further aspect of the present invention is the use of hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA in the preparation of a medicament for treating a proliferative disease.

A further aspect of the present invention is the use of an agent, a DNA template or a vector of the present invention for preparing a medicament for treating a proliferative disease.

A preferred use according to the present invention is a use as described above wherein the disease is selected from cancer such as a brain cancer including glioma, glioblastoma multiforme, medulloblastoma, astrocytoma, ependymonas, oligodendrogliomas, lung cancer, liver cancer, cancer of the spleen, kidney, adrenal gland (including pheochromocytoma), thyroid gland, oesophagus, pituitary gland, lymph node, small intestine, pancreas, blood (lymphomas and leukaemias), colon, stomach, breast, endometrium, prostate including prostate adenocarcinoma, testicle, ovary, skin including melanoma, head and neck, oesophagus and bone marrow.

A further aspect of the present invention is a method for diagnosing a proliferative disease by detecting the presence of hY1 RNA, hY3 RNA.U2 snRNA, 5S rRNA, hY4 RNA and hY5 RNA in a sample or tissue. This aspect derives from the finding that hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and hY5 RNA are required for initiation of DNA replication. And as it is known to the skilled person the level of initiation of DNA replication is particularly high in highly proliferative tissue, such as cancerous tissue. Thus hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and hY5 RNAs may serve as a marker for proliferate diseases. Therefore, agents capable of binding to hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and/or hY5 RNA or their protein complexes may be useful in labelling of cells and tissues for diagnostic purpose. The binding agent could be a protein agent, such as an antibody, or it could be a nucleic acid, such as a RNA or DNA probes, whereby a DNA probe is preferred. The binding agent may be labelled to allow detection. For example, the agent can be indirectly labelled with a Biotin marker or it could be directly labelled with a fluorescence marker. For example, detection of hY1 RNA, hY3 RNA, hY4 RNA and/or hY5 RNA can be achieved by reverse transcription-polymerase chain reaction (RT-PCR) using primers that amplify a DNA copy of U2 snRNA, 5S rRNA, hY1 RNA, hY3 RNA, hY4 RNA and/or hY5 RNA to allow their detection. It should be noted that the various detection label for binding agents according to the present invention are well known in the art. The method according to the present invention may be performed in vitro or in vivo. For example the method may be performed in vitro on an extract or homogenate derived from a sample extracted from a patient. Alternatively, the method may be performed on whole or fixed cells. Furthermore, the method may be performed directly in vivo, wherein the binding agent is administered into the patient and may label the highly proliferative tissue. A further aspect of the present invention is a cell free assay system for investigating DNA replication initiation, comprising

(a) cytosol from proliferative cells depleted of hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and hY5 RNA (b) and G1 phase nuclei

(c) NTPs and dNTPs

(d) hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and hY5 RNAs.

It will be appreciated by that the cell free assay system according to the present invention is an improvement of an existing cell free assay system described in US patent No. 6,107,042 and EP patent EP 0932664 B1. The difference to this cell free system is that the system according to the present invention comprises a cytosol fraction which is specifically depleted of hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and hY5 RNA. This specifically depleted fraction allows a more detailed study of the initiation of DNA replication. This advantageous depletion may be achieved by various means. Preferably, the depletion is achieved by fractionating the cell extract as described in figure 1 (A), wherein the QB fraction is purified over an Arginine Sepharose column at pH 8.2, wherein the bound RNAs are eluted at a pH of 8.2 to 9.0 preferably of 8.2 to 8.6. However, the depletion may also be achieved by other means well known in the art for example by enzymatic degradation of the RNAs e.g. by Ribonuclease A (whereby the enzyme is removed prior to use in the cell free assay according to the present invention) or by other known methods such as immunodepletion, or by antisense DNA- directed degradation of the said RNAs by using Ribonuclease H.

A further aspect of the present invention is a method of screening for an agent that modulates the chromosomal DNA replication, comprising

(a) Incubating a test agent in a cell free assay system according to the present invention;

(b) assessing the rate of initiation of chromosomal DNA replication in the composition, wherein a change in the rate of initiation of chromosomal DNA replication in the presence of the test agent indicates that the test agent may modulate DNA replication.

It will be appreciated that the ways to asses the rate of initiation of DNA replication are numerous and well known in the art. For example by detecting directly the DNA synthesis rate by e.g. labelling the dNTPs and/or NTPs and determining their incorporation into the DNA. Suitable labels include radioactive labels and fluorescence labels. The test agent which may be used may be of natural or synthetic origin for example, the test compound may be a small synthetic chemical compound as used in conventional drug screening programs, alternatively the compound may be from a natural source e.g. a cell extract from a prokaryote or eukaryote such as a plant or animal.

Furthermore, the test compound may have been designed by using hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and hY5 RNA as a model.

In a preferred aspect of the present invention hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and hY5 RNA are used to design oligonucleotides which either mimic or antagonise hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and hY5 RNA function. In particular, hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and hY5 RNA sequences or hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and hY5 RNA gene sequences can be used for this aspect of the invention.

A preferred aspect of the present invention is a method of designing, screening or identifying an agent which inhibits initiation of DNA replication comprising determining the three dimensional structure of hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and hY5 RNA and modelling the structure of the test compound according to the physical properties of hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and hY5 RNA, respectively. Such modelling techniques are well known in the art and employ known techniques of computational analysis and comparison. A further aspect of the present invention is a method for identifying initiation factors of chromosomal DNA replication comprising contacting an RNA selected from the group of hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA with cytosolic extract or a fraction thereof under physiological conditions and identify the cellular components binding to the RNA.

FIGURES

Figure. 1 : Isolation and identification of small soluble RNA as initiation factors for human chromosomal DNA replication. (A) Purification scheme. (B) Visualisation of the purified RNA species from the eluate fraction of the Arginine Sepharose column (fraction ArE) by agarose gel electrophoresis and staining with ethidium bromide. A 100bp DNA ladder was used as electrophoretic marker. (C) Reconstitution of initiation of human chromosomal DNA replication in vitro (29) with fractions described in panel A. Percentages of replicating nuclei were quantitated and mean values from two independent experiments are shown.

Figure. 2: Purification of small soluble RNA species. The RNA shown in Fig. 1 B was fractionated on a Superdex-200 gel filtration column. (A) Visualisation of the RNA-containing fractions by agarose gel electrophoresis and staining with ethidium bromide. A 100bp DNA ladder was used as electrophoretic marker. (B) Reconstitution of initiation of chromosomal DNA replication in vitro with fractions described in panel A. Percentages of replicating nuclei were quantitated and mean values from two independent experiments are shown. (C) Identification of candidate RNA species by Northern blots. The RNA species in the active fractions were identified by cDNA cloning and sequencing, as described in the main text. Radioactive dsDNA probes of these sequences were generated by PCR. Fractions of the Superdex-200 gel filtration shown in panel A were blotted onto Nylon membranes, which were then hybridised with the indicated probes. The asterisk on the U2 snRNA panel denotes a bleed-through signal from the 5S rRNA sample because this latter membrane was stripped and re-hybridised with the U2 probe. The arrowheads indicate discernible RNA species detected by the specific probes.

Figure. 3 Reconstitution of initiation of chromosomal DNA replication with in- vitro-transcribed recombinant RNA. The indicated full-length RNA species were cloned in prokaryotic transcription vectors under the control of the bacteriophage SP6 promoter. Recombinant RNA species were generated by in-vitro-transcription, purified and 50ng of each recombinant RNA species was functionally tested in the human cell-free DNA replication initiation system (29) in the presence of fractions QA and ArFT. One microgram of unfractionated RNA purified from fraction ArE was used as positive control. Percentages of replicating nuclei were quantitated and mean values from two independent experiments are shown.

Figure 4.: Represents the complete sequence of genomic DNA of the human autoantigen hY5 Ro RNA gene (hY5)

Figure 5. Represents the genomic DNA of the human small cytoplasmic Y RNA gene (hY4).

Figure 6. Represents the genomic DNA of the human 5S ribosomal RNA gene (5S).

Figure 7. Represents the genomic DNA of the human gene for small nuclear RNA U2 (snRNA U2).

Figure 8. Figure 8A. represents the full-length sequences of the RNA species coded by the hY4 gene (the code is written in DNA form (substitute T for U for the RNA code).

Figure 8B. represents the full-length sequences of the RNA species coded by the hY5 gene (the code is written in DNA form (substitute T for U for the RNA code). Figure 8C. represents the full-length sequences of the RNA species coded by the 5S gene (the code is written in DNA form (substitute T for U for the RNA code).

Figure 8D. represents the full-length sequences of the RNA species coded by the U2 gene (the code is written in DNA form (substitute T for U for the RNA code).

Figure 9. Fig. 9 A-C represents hY5 siRNA structures which were generated using Ambion's siRNA Target Finder engine using siRNA design guidelines described by Elbashir et al. (Genes Dev. 2001 Jan 15; 15(2): 188-200). The siRNAs may be generated with tt/uu 3'overhangs on the duplex, or may have these incorporated by the vector system used to introduce them to the cell (Brummelkamp, T. et al. Science. 2002 Apr 19;296(5567):550-3. Epub 2002 Mar 21).

Figure 10. Fig. 10 A-C represents hY4 siRNA structures which were generated using Ambion's siRNA Target Finder engine using siRNA design guidelines described by Elbashir et al. (Genes Dev. 2001 Jan 15;15(2):188- 200). The siRNAs may be generated with tt uu 3'overhangs on the duplex, or may have these incorporated by the vector system used to introduce them to the cell (Brummelkamp, T. et al. Science. 2002 Apr 19;296(5567):550-3. Epub 2002 Mar 21).

Figure 11. Fig. 11 A-D represents 5S siRNA structures which were generated using Ambion's siRNA Target Finder engine using siRNA design guidelines described by Elbashir et al. (Genes Dev. 2001 Jan 15;15(2): 188-200). The siRNAs may be generated with tt/uu 3'overhangs on the duplex, or may have these incorporated by the vector system used to introduce them to the cell (Brummelkamp, T. et al. Science. 2002 Apr 19;296(5567):550-3. Epub 2002 Mar 21).

Figure 12. Fig. 12 A-F represents U2 siRNA structures which were generated using Ambion's siRNA Target Finder engine using siRNA design guidelines described by Elbashir et al. (Genes Dev. 2001 Jan 15; 15(2): 188-200) ). The siRNAs may be generated with tt/uu 3'overhangs on the duplex, or may have these incorporated by the vector system used to introduce them to the cell (Brummelkamp, T. et al. Science. 2002 Apr 19;296(5567):550-3. Epub 2002 Mar 21).

Figure 13. Fig. 13 represents 65 predicted sequences of Antisense oligonucleotides which may be used to bind hY5 RNA. The sequences were generated using AOPredict, an Antisense oligonucleotide prediction program (Chalk AM, Sonnhammer EL. Genomic gene clustering analysis of pathways in eukaryotes. Genome Res. 2003 May;13(5):875-82. Epub 2003 Apr 14.)

Figure 14. Fig. 14 represents 76 predicted sequences of Antisense oligonucleotides which may be used to bind hY4 RNA. The sequences were generated using AOPredict, an Antisense oligonucleotide prediction program (Chalk AM, Sonnhammer EL. Genomic gene clustering analysis of pathways in eukaryotes. Genome Res. 2003 May;13(5):875-82. Epub 2003 Apr 14.)

Figure 15. Fig. 15 represents 102 predicted sequences of Antisense oligonucleotides which may be used to bind 5S RNA. The sequences were generated using AOPredict, an Antisense oligonucleotide prediction program (Chalk AM, Sonnhammer EL. Genomic gene clustering analysis of pathways in eukaryotes. Genome Res. 2003 May;13(5):875-82. Epub 2003 Apr 14.)

Figure 16. represents 164 predicted sequences of Antisense oligonucleotides which may be used to bind U2 RNA. The sequences were generated using AOPredict, an Antisense oligonucleotide prediction program (Chalk AM, Sonnhammer EL. Genomic gene clustering analysis of pathways in eukaryotes. Genome Res. 2003 May;13(5):875-82. Epub 2003 Apr 14.)

Figure 17. Fig 17 represents the genomic DNA of the human hY1 RNA gene.

Figure 18. Fig 18 represents the genomic DNA of the human hY3 RNA gene. Figure 19. Figure 19A represents the full-length sequences of the RNA species coded by the hY1 gene (the code is written in DNA form (substitute T for U for the RNA code).

Figure 19B represents the full-length sequences of the RNA species coded by the hY3 RNA gene (the code is written in DNA form (substitute T for U for the RNA code).

Figure 20. Figure 20 represents hY1 siRNA structures which were generated using Ambion's siRNA Target Finder engine using siRNA design guidelines described by Elbashir et al. (Genes Dev. 2001 Jan 15;15(2): 188-200). The siRNAs may be generated with tt/uu 3'overhangs on the duplex, or may have these incorporated by the vector system used to introduce them to the cell (Brummelkamp, T. et al. Science. 2002 Apr 19;296(5567):550-3. Epub 2002 Mar 21).

Figure 21. Figure 21 represents hY3 siRNA structures which were generated using Ambion's siRNA Target Finder engine using siRNA design guidelines described by Elbashir et al. (Genes Dev. 2001 Jan 15;15(2):188-200). The siRNAs may be generated with tt/uu 3'overhangs on the duplex, or may have these incorporated by the vector system used to introduce them to the cell (Brummelkamp, T. et al. Science. 2002 Apr 19;296(5567):550-3. Epub 2002 Mar 21).

Figure 22. Figure 22 represents 148 predicted sequences of Antisense oligonucleotides which may be used to bind hY1 RNA. The sequences were generated using AOPredict, an Antisense oligonucleotide prediction program (Chalk AM, Sonnhammer EL. Genomic gene clustering analysis of pathways in eukaryotes. Genome Res. 2003 May;13(5):875-82. Epub 2003 Apr 14.).

Figure 23. Figure 23 represents 153 predicted sequences of Antisense oligonucleotides which may be used to bind hY3 RNA. The sequences were generated using AOPredict, an Antisense oligonucleotide prediction program (Chalk AM, Sonnhammer EL. Genomic gene clustering analysis of pathways in eukaryotes. Genome Res. 2003 May;13(5):875-82. Epub 2003 Apr 14.).

Figure 24. Figure 24 A. Human Y RNA is required for initiation of chromosomal DNA replication. In-vitro synthesis of full-length human wild- type RNA. The indicated RNA species were synthesised by the SP6 bacteriophage system.

Figure 24B. Functional reconstitution of chromosomal DNA replication with hY RNA. Nuclei from mimosine-arrested HeLa cells were incubated in fractions QA and ArFT, supplemented with 100nM of the individual RNA species synthesised in vitro as indicated.

Figure 25 represents nucleotide sequences and predicted secondary structures of human Y RNAs. (Nucleotide sequences and predicted structures for hY1 , hY3, hY4 and hY5 RNA were adapted from O'Brien et al., 1993; van Gelder et al., 1994, references 96-97). The nucleotide sequences complementary to small interfering RNAs (siRNAs) that were synthesised for use in RNA interference experiments (RNAi) are shown in bold.

Figure 26. HeLa cells were transfected with siRNAs directed against hY1 RNA alone, with a mixture of siRNAs directed against all four hY RNAs together and, as negative control, with siRNA directed against the non-human luciferase gene. Figure 26 A. Flow cytometry of cell nuclei isolated at 48h after transfection. Analysis of nuclear DNA content by flow cytometry shows an increased proportion of G1 phase nuclei with a concomitant decrease of S phase nuclei upon transfection with siRNA against the hY RNA loop domains. In contrast, no significant change in cell cycle phase proportions was obtained upon transfection with siRNAs directed against luciferase mRNA.

Figure 26 B. Depletion of either hY1 alone, or all four hY RNAs together resulted in the significant reduction of S phase nuclei in the preparation. At 47h post transfection the cells were pulse labelled for 1 h with BrdU and the percentages of BrdU-positive S phase cells were determined by immunofluorescence microscopy.

Figure 27. Predicted secondary structures of human Y RNAs. Nucleotide sequences and predicted structures for hY1 , hY3, hY4 and hY5 RNA were adapted from (O'Brien et al., 1993; van Gelder et al., 1994). Nucleotide sequences complementary to antisense DNA oligonucleotides are in bold.

Figure 28. Inhibition of initiation by antisense DNA oligonucleotides complementary to hY RNA loop domains. Nuclei from mimosine-arrested HeLa cells were incubated in S100 cytosolic extract, supplemented with the indicated antisense DNA oligonucleotides at the indicated final concentrations. When added in increasing amounts to reactions containing G1 phase template nuclei and unfractionated cytosolic extract, all four loop-specific antisense oligos significantly decreased the percentages of replicating nuclei in a dose-dependent manner. Mean values and standard deviations of 3-6 independent experiments (n=3-6) are shown.

EXAMPLE

Purification of hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and hY5 RNA from Cytosolic HeLa extract from a proliferative cell population

Cell culture and synchronisation

Human HeLa S3 and EJ30 cells were cultured as monolayers in Dulbecco's MEM plus 10% foetal bovine serum, 10 U/ml penicillin and 0. 1 mg/ml streptomycin (all from Gibco), and synchronised in early S phase exactly as described (28). Cells were arrested in late G1 phase by treatment with 0. 5 mM mimosine (Sigma) for 24 h (34). All cell synchronisations were verified by flow cytometry of isolated nuclei (28).

Preparation of nuclei and cell extracts Nuclei were prepared by hypotonic treatment of the cells, followed by Dounce homogenisation as described (28). Nuclei were stored in liquid N2 without loss of replication competence (29). Cytosolic and nuclear extracts were prepared as described (28).

Protein purification

All steps were carried out at 4°C. Crude cytosolic HeLa extract was obtained from 4C Biotech (Seneffe, Belgium), thawed on ice and was clarified by ultracentrifugation for 1 h at 100 000 g. This 100 000 g supernatant (S100) was loaded onto a Q Sepharose Hi-Load 26/10 column in an AktaFPLC system (Amersham Pharmacia Biotech), pre-equilibrated in buffer A (50 mM Tris-HCI pH 7. 8, 1 mM DTT, 1 mM EGTA) containing 200 mM KCI. The protein flowing through the column was collected and the column was eluted sequentially with 280 and 500 mM KCI in buffer A. The resulting protein fractions were termed QFT, QA and QB, respectively.

hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and hY5 RNA were purified by fractionating the fraction termed QB (83) into two more fractions on Arginine Sepharose, followed by a purification of the 0. 35M eluate, termed ArE.

Arginine Sepharose Column

Fraction QB (83) was diluted 1 :3 in 50mM Tris-HCI at pH 8.2, 1mM EGTA, 1mM DTT to adjust the KCI concentration to about 150mM, and subsequently loaded onto Arginine-Sepharose (Amersham Biosciences) equilibrated in 150mM KCI, 50mM Tris-HCI pH 8.2, 1mM EGTA, 1mM DTT. The column was extensively washed in this equilibration buffer. Bound RNA (and residual protein) was eluted in 350mM KCI, 50mM Tris-HCI at the pH range of 8.2-8.6, 1mM EGTA, 1 mM DTT. The RNA was further purified by phenol/chloroform extraction and precipitated in ethanol (which is a standard technique). Gel filtration

The precipitated RNA was dissolved in 10mM Tris-HCI pH 8, 1mM EDTA (TE buffer) and loaded on a Superdex-200 column (Amersham Biosciences) equilibrated in TE buffer. RNA was fractionated on this column according to size under standard chromatography conditions. Individual fractions were concentrated by ethanol precipitation and the RNA contained in each was analysed by agarose gel electrophoresis and visualised by staining with ethidium bromide.

Functional testing of the eluated fractions indicated that QB contains at least two independent initiation factors, one present in the 0. 15M flow-through and one present in the eluate. Both need to be combined in the further presence of fraction QA (83) to reconstitute initiation of human chromosomal DNA replication in late G1 phase nuclei. Fraction ArE did not contain detectable levels of protein, but significant amounts of small RNA molecules. We purified these RNA molecules by phenol extraction and ethanol precipitation from ArE and showed in reconstitution experiments that they were the active component of ArE. We fractionated these RNA species further on the basis of size by gel filtration on Superdex 200. The initiation-promoting RNA co- fractionated with fractions #40-48, establishing that it is discrete species (or a collection of species) of RNA that is selectively promoting initiation. We characterized the initiation-promoting RNA by cDNA synthesis using this initiation-promoting RNA as template for reverse transcription and second strand synthesis, followed by cloning, DNA sequencing and BLAST analysis. Using unfractionated RNA as template from either QB or ArE, many discrete species of small human RNA molecules were identified, all through multiple isolates. Using the size-fractionated RNA of gel-filtration fractions #40-48 as template, we identified only four discrete species, again in multiple isolates: U2 snRNA, 5S rRNA, hY4 RNA, and hY5 RNA. Northern blot analysis showed that hY1 and hY3 RNA were also present in these active fractions in addition to U2 snRNA, 5S rRNA, hY4 RNA, and hY5 RNA, but in reduced amounts compared to these latter RNAs. Furthermore, we generated in vitro transcribed versions of these RNA species for defined reconstitution experiments to determine the identity/identities of the active initiation- promoting RNA. Purified in vitro transcribed full length hY1 RNA, hY3 RNA, hY4 RNA, and hY5 RNA were able to substitute for the active preparation of initiating RNAs from fraction ArE to initiate chromosomal DNA replication in late G1 phase template nuclei, establishing that these RNA constitute the active component.

DNA replication reactions and analysis of reaction products

DNA replication reactions contained the following: a buffered mixture of NTPs and dNTPs (elongation buffer; 29), including 150 pmol digoxigenin-dUTP (Roche) as a probe for microscopic detection, 100 μg HeLa unfractionated cytosolic extract or variable amounts of fractions from the protein purification as specified in the figure legends, and 2-5 x 105 late G1 phase nuclei. The reaction volume was adjusted to 50 μl with buffer B containing 100 mM K- acetate (see above). All fractions from the protein purification were first dialysed against this buffer before addition to the reaction. Incubation time was 3 h, unless indicated otherwise.

Detection of DNA replication by confocal microscopy was performed exactly as described (29), except that anti-digoxigenin fluorescein-labelled Fab fragments (Roche) were used at 1 :100 dilution for the staining of newly replicated DNA. Nuclear DNA was counterstained with propidium iodide. Stained nuclei were analysed using a Leica confocal laser microscope as specified (30).

RNA interference

siRNAs were chemically synthesised using an Ambion Silencer^® siRNA construction kit according to the instructions of the manufacturer. The following DNA oligonuceotides were synthesised (Sigma-Genosys) to direct generation of siRNAs against the following RNAs in vitro: Luciferase mRNA: Lud , 5'-AACTTACGCTGAGTACTTCGACCTGTCTC; Luc2, 5'- AATCGAAGTACTCAGCGTAAGCCTGTCTC; hY1 RNA: hY1A, 5'- TGTTCTACTCTTTCCCCCCTTCCTGTCTC; hY1 B, 5'- AAGGGGGGAAAGAGTAGAACACCTGTCTC; hY3 RNA: HY3A, 5'- TTCTTTGTTCCTTCTCCACTCCCTGTCTC; HY3B, 5'- GAGTGGAGAAGGAACAAAGAACCTGTCTC; hY4 RNA: HY4A, 5'- ACTAAAGTTGGTATACAACCCCCTGTCTC; HY4B, 5'-

GGGTTGTATACCAACTTTAGTCCTGTCTC; hY5 RNA: HY5A, 5'- GTTGATTTAACATTGTCTCCCCCTGTCTC; HY5B, 5'- GGGAGACAATGTTAAATCAACCCTGTCTC. Working stock solutions of siRNA were prepared at a concentration of 1 μM by diluting the main siRNA stock solution with resuspension buffer (0.2μm-filtered sterile RNase-free water, 100mM NaCI, 50mM Tris-HCI pH7.5) in a sterile RNase-free microcentrifuge tube.

Transfections were performed with the 7ranslT^®-TKO transfection reagent (Mirus) on 6-well plates essentially as specified by the manufacturer. At 24h prior to transfection, 1.2 x 10⁵ HeLa S3 cells were seeded per well for a 24- well plate. The final concentration of siRNA in the culture medium was either 3nM or 10nM, yielding identical results. Inhibition of initiation by antisense DNA oligonucleotides Template nuclei and cytosolic extracts were prepared as previously detailed (29, 83). DNA replication reactions were performed exactly as described (83); and references therein).

Cytosolic extracts were pretreated, where indicated, with a final concentration of 0.3mg/ml ribonuclease A (RNAse A, Roche) at room temperature for 1-2h, and then used immediately in DNA replication reactions as detailed above. The following single-stranded DNA oligonucleotides were synthesised (Sigma- Genosys): T3 sequencing primer, 5'-CGAAATTAACCCTCACTAAAGGGA; conserved hY stem, 5'-CNNTCGGACCAGCC; hY1 loop, 5'- AAGGGGGGAAAGAGTAGAACAAGGA; hY3 loop, 5'- GAGTGGAGAAGGAACAAAGAAATCT; hY4 loop, 5'- GGGTTGTATACCAACTTTAGTGACA; hY5 loop, 5'-

GGGAGACAATGTTAAATCAACTTAA. Their positions on the complementary hY RNAs are highlighted in Figure 5A. These antisense DNA oligonucleotides were added directly to DNA replication reactions at the indicated final concentration. They were stored in aliquots at -80°C in distilled water. Detection of DNA replication by confocal microscopy was performed exactly as described (83); and references therein). Quantitation of DNA synthesis was done by incorporation of α³²P-dCTP followed by precipitation with tri-chloro- acetic acid and scintillation counting as detailed previously (28).

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Claims

1. Agent that modulates chromosomal DNA replication by modulating the function of hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and/or hY5 RNA.

2. An agent according to Claim 1 that inhibits chromosomal DNA replication.

3. An agent according to Claim 1 that increases chromosomal DNA replication.

4. An agent according to any of Claims 1-2 wherein the agent is an antibody against hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA.

5. An agent according to Claims 1-2 wherein the agent inhibits expression of one or more genes selected from the group of hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and hY5 RNA.

6. An agent according to Claim 3 wherein the agent stimulates expression of one or more genes selected from the group of hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and hY5 RNA.

7. An agent according to Claim 5 wherein the agent comprises a antisense RNA, a small interfering RNA, or an engineered transcription repressor that inhibits chromosomal DNA replication gene transcription.

8. An agent according to Claim 7 wherein the agent is an RNA comprising a double stranded structure which is substantially identical to at least part of a target gene selected from the group of hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and hY5 RNA.

9. A DNA template encoding the RNA according to claim 8.

10. A vector comprising the DNA template according to claims 9.

11.A pharmaceutical preparation comprising an agent according to any of claims 1 - 8, a DNA template according to claim 9 or a vector according to claim 10.

12. Use hY1 RNA, hY3 RNA, of U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA in the diagnosis of a proliferative disease.

13. Use of an inhibitor of hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA mediated initiation of chromosomal DNA replication for the preparation of a medicament for treating a proliferative disease.

14. Use of hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA in the preparation of a medicament for treating a proliferative disease.

15. Use of an agent according to any of claims 1 - 8, a DNA template according to claim 9 or a vector according to claim 10 for preparing a medicament for treating a proliferative disease.

16. Use according to any of Claims 13 - 15 wherein the disease is selected from cancer such as a brain cancer including glioma, glioblastoma multiforme, medulloblastoma, astrocytoma, ependymonas, oligodendrogliomas, lung cancer, liver cancer, cancer of the spleen, kidney, adrenal gland (including pheochromocytoma), thyroid gland, oesophagus, pituitary gland, lymph node, small intestine, pancreas, blood (lymphomas and leukaemias), colon, stomach, breast, endometrium, prostate including prostate adenocarcinoma, testicle, ovary, skin including melanoma, head and neck, oesophagus and bone marrow.

17. Method for diagnosing a proliferative disease comprising detecting the presence of hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and hY5 RNA in a sample or tissue.

18. A cell free assay system for investigating DNA replication initiation, comprising (a) cytosol from proliferative cells depleted of hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and/or hY5 RNA (b) and G1 phase nuclei (c) NTPs and dNTPs (d) hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA and/or hY5 RNAs.

19. A method of screening for an agent that modulates the chromosomal DNA replication, comprising

(a) Incubating a test agent in a cell free assay system according to claim 18; (b) assessing the rate of initiation of chromosomal DNA replication in the composition, wherein a change in the rate of initiation of chromosomal DNA replication in the presence of the test agent indicates that the test agent may modulate DNA replication.

20. A method for identifying initiation factors of chromosomal DNA replication comprising contacting an RNA selected from the group of hY1 RNA, hY3 RNA, U2 snRNA, 5S rRNA, hY4 RNA or hY5 RNA with cytosolic extract or a fraction thereof under physiological conditions and identify the cellular components binding to the RNA.