Nucleic acid binding peptide

Field of the invention

[0001] The present invention relates to means and methods for regulating gene expression. Specifically the invention relates to methods and uses of phospholipids for detaching zinc finger proteins from chromatin/DNA. Furthermore, the invention relates to a method for nucleic acid binding.

Background of the invention

[0002] Protein-nucleic acid recognition is centra! to large number of biomolecular control mechanisms, which regulate the functioning of eukaryotic and prokaryotic cells. For instance, protein-DNA interactions form basis for the regulation of gene expression. Many DNA binding proteins contain independently folded domains for the recognition of DNA, and these domains in turn belong to a large number of structural families such as leucine zipper, the "helix- turn-helix" and zinc finger families.

[0003] Zinc fingers take their name from the finger-shaped structure in which small group of conserved amino acids bind a zinc ion and forms a relatively independent domain in the protein. Zinc finger modules are approximately 30 amino acid-long motifs found in a wide variety of transcription regulatory proteins in eukaryotic organisms. Most zinc finger proteins are thought to act as DNA-binding regulators of transcription, however they are now known to bind RNA, protein and lipid substrates as well (Brown, R. S., (2005) Curr Opin Struct Biof, 15:94-8, Matthews J. M. and Sonde M., (2002) IUBMB Life VoI 54, 6: 351-5).

[0004] The most common form of the zinc finger motif, C2H2-type, was first described as a repeated domain in the Xenopus transcription factor TFIIIA (Miller et al., (1985) EMBO J. 4:1609-1614). C2H2-type zinc finger motifs share a common backbone polypeptide sequence of Cys-X ₂ - ₄ -Cys-X ₃ -Phe- X ₅ -Leu-X ₂ -His-X3-His, where x can be any amino acid. The conserved structural features are the tetrahedral coordination of zinc by two Cys and two His, and the presence of an antiparallei β-hairpin followed by α-helix, the principal recognition element responsible for specific binding to a nucleotide triplet. Zinc fingers are autonomous folding units: an isolated zinc finger folds into a compact domain. Hence, this motif is highly versatile. Extended DNA sequences can be specifically recognized simply by bringing together a series of zinc fingers that have different residues in their α-helices. The strength and specificity

of the DNA interaction is adjusted by changes in the number of zinc finger repeats. Thus, the arrays of zinc fingers are well suited for combinatorial recognition of DNA sequences.

[0005] Zinc finger motifs may be found dispersed throughout a polypeptide or may be clustered into one or more tandemly repeated blocks. Zinc finger motifs recur in large number of transcription factors. Zinc finger motif containing proteins have been implicated in a wide range of biological activities that includes e.g. regulation of cell proliferation and differentiation as we!l as controlling patterns of embryonic development.

[0006] Zinc finger proteins, i.e. proteins comprising a zinc finger motif, have been used in a variety of isolation methods e.g. in isolation of plasmid- DNA (Woodgate et al., (2002) Biotechnology and bioengineering VoI 79:4 p.450-456) and detection of PCR products (Osawa et al., (2008) Nucleic Acid Symposium series No 52:23-24).

[0007] The zinc finger protein's ability to recognize a wide variety of DNA sequences has made it an attractive framework to design novel DNA- binding peptides/proteins. However, there remains a need in the art for the development of nucleic acid binding proteins without any sequence specificity. In addition novel functional domains useful in design of zinc finger transcription factor-based technologies are needed. Although zinc finger proteins and motifs have been extensively studied, a little is known about the ways of regulating their nucleic acid binding in vivo. Thus, novel means for regulating the interaction of zinc fingers and their target promoter in the chromatin are needed.

[0008] Furthermore, zinc finger proteins are known to regulate gene expression by recruiting histone-modifying enzymes, such as histone deacety- lases (HDACs), to the promoter region of a target gene. Deacetyiation (removal of acetyl groups) of histones results in condensation of chromatin structure thereby preventing gene transcription. In many cancers, tumor suppressor genes are silenced by this mechanism.

£0009] There have been attempts to develop HDAC inhibitors for cancer therapy. Many clinical trials are ongoing and some FDA approved inhibitors are already available. HDAC inhibitors have also been used in AIDS therapy for eliminating latent human immunodeficiency viruses (HIV). Latently infected cells are a permanent source of infectious particles even after long periods of antiviral therapy. Forced expression of HiV genes by HDAC inhibitors

may together with antiviral therapy lead to elimination of the latently expressed cells and thus to the cure of the infection.

[0010] However, current HDAC inhibitors are rather unspecific as it is known that HDACs deacetyiate also other proteins than histones and thereby their inhibition can cause many undesirable side effects. Thus, more specific means and methods for controlling the recruitment of HDACs by zing finger proteins and for regulating gene expression are needed. Especially, there is a need in the art for activating tumor suppressor genes and removing latent HIV reservoirs.

Brief description of the invention

[0011] The present invention is based on a study aimed to characterize SAP30L and SAP30 proteins, and on a finding that said proteins have intrinsic nucleic acid binding activity, which is partly mediated through a novel N-terminal Zinc-containing structure. Surprisingly, it was also found that the nucleic acid binding can be regulated by phospholipids.

[0012] An object of the present invention is to provide novel means and methods for regulating gene expression, especially in treating cancer or viral infections such as HiV infection, Rift Valley fever, and influenzas such as avian flu and swine flu.

[0013] Another object of the present invention is to provide noveϊ means and methods useful in nucieic acid binding and regulation of gene expression so as to fulfil the above needs, in particular, an object of the present invention is to provide novel means and methods for nucleic acid binding without any sequence specificity.

[0014] A further object of the invention is to provide novel means and methods for nucleic acid binding, wherein the nucleic acid binding can be regulated.

[0015] Still a further object is to provide a method for binding nucleic acids for use e.g. in DNA isolation, purification, concentration and transfer.

[0016] The present invention relates to a use of a phospholipid for detaching a zinc finger protein from a chromatin or DNA and to a method for said detachment by said phospholipids.

[0017] The present invention also relates to a use of a phospholipid for the manufacture of a medicament for use in zinc finger therapy. In some embodiment said therapy is used for treating cancer or a viral infection by de-

taching a zinc finger protein from a chromatin or DNA. In more specific embodiments, the viral infection to be treated by the zinc finger therapy is selected from a group consisting of HiV infection, Rift Valley fever and influenza including but not limited to avian flu and swine flu.

[0018] The present invention further relates to a method for binding nucleic acids, which method comprises:

[0019] 1) adding a zinc finger protein or peptide to a nucleic acid solution

[0020] 2) forming a peptide-nucieic acid complex

[0021] 3) adding a phospholipid to release the nucleic acid from the peptide-nucieic acid complex.

[0022] The present invention also relates to a use of a protein having a sequence set forth in SEQ ID NO: 1 or a protein having at least 70%, preferably at least 90%, most preferably at least 95% sequence identity to SEQ ID NO: 1 , or a variant or a fragment thereof, for nucleic acid binding. Likewise, the present invention also relates to a use of a protein having a sequence set forth in SEQ ID NO: 3 or a protein having at least 70%, preferably at ieast 90%, most preferably at least 95% sequence identity to SEQ ID NO: 3, or a variant or a fragment thereof, for nucleic acid binding.

[0023] Specific embodiments of the invention are set forth in the dependent claims.

[0024] Other objects, details and advantages of the present invention will become apparent from the following drawings, detailed description and examples.

Brief description of the drawings

[0025] in the following the invention will be described in greater detail by means of preferred embodiments with reference to the attached drawings, in which

[0026] Figure 1 shows a DNA ladder electrophoretic mobility shift assay (L-EMSA) with the GST-SAP30/SAP30L fusion proteins.

[0027] Figure 2 shows a L-EMSA with the GST-SAP30L 1-92 fusion protein in the presence of 50 mM 1 , 10-o-phenanthroline, a zinc chelating agent.

[0028] Figure 3A shows a lipid biot assay, in which the relative affinities of GST-SAP30L and GST-SAP30 for various PIs were quantified.

[0029] Figure 3B shows a protein-lipid plot assay, where GST fusion proteins GST-SAP30L (1-92) and GST-SAP30L (78-92) (0.5 μg/ml) were incubated with PlP strips. The lipids which bound most strongly are indicated.

[0030] Figure 4 shows a L-EMSA with GST-SAP30L 1-92 in the presence of equivalent molar quantities of Ptdlns or Ptdlns(5)P.

[0031] Figure 5 shows a conventional EMSA assay with GST- SAP30L 1-92 and 5S ribosomal RNA probe in the presence of equivalent molar quantities of Ptdlns or Ptdlns(5)P.

Detailed description of the invention

[0032] The present invention is based on studies aimed to investigate the functions and the domain structure of SAP30L and SAP30 proteins. Human SAP30L (SAP30-like) (SEQ ID NO: I) ₅ thus named because it shares 70% sequence identity with SAP30 (Sin3A-associated p_rotein 30) (SEQ ID NO: 3), is the "newest" member of the Sin3A corepressor complex. It was originally discovered as an expressed transcript in cultured T84 cells induced to differentiate in response to TGF-β (Lindfors, K. (2003) BMC Genomics 4:53). Since then SAP30L has been shown to associate with the Sin3A-HDAC complex and to induce transcriptional repression in a Sin3A- and histone deacetylase-dependent manner (Viiri K. et al., (2006) Nucleic Acid Res 34:3288-98). SAP30L protein is 183 amino acids in length and located in the nucleus.

[0033] It has been found out that zinc finger proteins, including SAP30L and SAP30, bind DNA directly and interact with core histones in a chromatin structure. It has been further surprisingly found out that both DNA binding and chromatin association are regulated by phospholipids, such as phosphatidylinositols.

[0034] According to the invention, the nucleic acid binding of zinc finger proteins can be regulated by two ways. The binding can be regulated by zinc-cheiating agents, such as 1 ,10-o-phenanthroline or ethylenediaminetetraacetic acid (EDTA). Zinc chelating agent is applied to peptide-nucleic acid complex in a suitable concentration to release the nucleic acid from the complex. A preferably used zinc-chelating agent is 1 ,10-o- phenanthroline in a concentration of 10-10OmM. The peptide thus functions as a "zinc-switch" meaning that the DNA/RNA bound to the peptide can be released by zinc-chelating agents.

[0035] In another aspect the nucleic acid binding and chromatin association of zinc finger proteins is regulated by phospholipids. The zinc finger proteins function as "lipo-switchs" meaning that the DNA/RNA bound to the protein can be released by phospholipids. Examples of the phospholipids useful in the invention are phophatidyfinositols or derivatives thereof. Preferably the phosphatidylinositols (PIs) are mono- or diphosphorylated phosphoinositides (PIP), more preferably monophosphorylated phosphoinositides PI(3)P, PI(4)P or PI(5)P. One preferred phosphatidyϋnositol derivative is inositolphosphate. The interaction of the peptide with PIs is mediated by the phosphoinositide (Pl)-binding polybasic region ( ⁸⁵ RNKRKRK ⁹¹ ) and supported by the preceding hydrophobic region and the zinc-coordinating structure. The specificity of Pl binding is partially determined by the composition of the basic sequence of the phosphoinositide (Pl)-binding polybasic region. It is noteworthy that the same region of the zinc finger proteins may interact with both DNA/RNA and PIs.

[0036] The present invention provides a method of detaching zinc finger proteins from chromatin or DNA by phospholipids. This opens a whole new approach for regulating gene expression. When used for treating a disease or a medical condition, the method may be referred to as a zinc finger therapy. A particularly important embodiment of the present invention is cancer therapy by activating tumor suppressor genes by detaching zinc finger proteins from chromatin. The detachment of zinc finger proteins leads to abolishment of recruited HDACs thus allowing acetylation of histones, loosening of the chromatin structure and subsequent activation of tumor suppressor genes.

[0037] Detachment of zinc finger proteins from DNA or chromatin is a more specific way of activating gene expression than the use of current HDAC inhibitors. However, HDAC inhibitors may be used in combination with phospholipids to enhance the activation of gene transcription.

[0038] !n some embodiments, the zinc finger therapy according to the present invention may be used for treating vira! infections such as AIDS. The detachment of zinc finger proteins from chromatin by phospholipids leads to transcriptional activity as described above. In this way, HIV genes may be forced to be expressed by latently infected leukocytes. Normal antiviral therapy may then be applied for destroying infected cells. As a result, the latent HIV reservoirs may be permanently eliminated and the infection cured.

[0039] In other embodiments, the zinc finger therapy may be

directed to treating other viral infections such as Rift Valley fever and influenzas, including but not limited to avian flu or swine flu. Without being limiting to the present invention or its embodiments, it is hypothesized that viral nonstructural protein NSs interact with and recruit the host SAP30/HDAC complexes thereby inhibiting interferon β (INF-β) expression and consequently the host antiviral response. By the present zinc finger therapy it is possible to antagonize the inhibition of INF-β expression and elicit the antiviral response of the host cells.

[0040] The above aspects of the present invention may be formulated such that the invention relates to the use of phospholipids for detaching zinc finger proteins from chromatin/DNA. Thus, all embodiments of the present method apply for the medicinal use of the phospholipids.

[0041] The present invention also provides a method for nucleic acid binding, wherein a zinc finger protein or peptide having an ability to bind nucleic acids and phospholipids is contacted with a nucleic acid in a solution to form a protein-nucleic acid complex. The nucleic acid may then be released from the complex by a phospholipid or a zinc chelating agent. One preferred peptide for use in the method for nucleic acid binding comprises a fragment of SAP30L having an ability to bind nucleic acids and phospholipids, or a variant thereof having the same ability.

[0042] Herein, the term "peptide" refers to two or more amino acids joined together by peptide bonds. The peptide for use in the present invention has nucleic acid binding activity, can be regulated by phospholipids and can be of different length from less than 20 to over 100 amino acids long. Preferably the peptide contains 14 to 115 amino acids and comprises amino acids 1 to 92 of the SEQ ID NO: 1 (SAP30L) or corresponding amino acids of SEQ ID NO: 3 (SAP30). More preferably the peptide contains 14 to 115 amino acids and comprises amino acids 78 to 92 of the SEQ ID NO: 1 (SAP30L) or corresponding amino acids of SEQ ID NO: 3 (SAP30). Most preferably the peptide contains amino acids 78 to 92 of the SEQ ID NO: 1 (SAP30L) or corresponding amino acids of SEQ ID NO: 3 (SAP30).

[0043] The term "variant" of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 include alterations to the protein such as a change, loss or addition of an amino acid, truncation or fragmentation of the protein or increase in the size of the protein. Alterations can increase degradation of the protein, can decrease degradation of the protein or can change conformation

of the protein. However, the variants retain the function, i.e. the zinc-switch and lipo-switch activity, of the protein or peptide. The alteration need not be in an active site of the protein, but may be present in the functional domain. Alterations can include modifications to the protein such as phosphorylation, myristy- iation, acetylation or methylation. Functional domain in this context thus refers to nucleic acid, zinc or phospholipid binding domain.

[0044] The term "fragment thereof as used herein refers to subre- gions of a sequence which still retain the function of the peptide. Fragments may be any region or regions of said sequence.

[0045] Peptides for use in the invention can be produced by any suitable method in the art. They can e.g. be synthesized chemically or isolated from a protein hydrolysate. in one embodiment of the invention the peptides are conveniently produced by recombinant technology. This denotes isolation of a nucleotide sequence by ampiification in a PCR reaction (Ausubel et at., 1989) or other generally known recombinant techniques (Sambrook et al., 1989), insertion of the sequence under a strong promoter in an expression vector, transfer of the vector in suitable host cells, cultivation of the host cells and recovering the peptide from the culture.

[0046] Expression vector as used herein refers to a cloning plasmid or vector capable of expressing DNA encoding the peptide described herein after transformation into a desired host. Desirably, a recombinant vector is constructed in which the polynucleotide sequence is operably linked to a heterologous expression control sequence permitting expression of the protein. Numerous types of appropriate expression vectors are available and selection of appropriate vector will depend on the intended use of the vector, and the host cell transformed with the vector.

[0047] It is desirable to express a peptide suitable for use in the invention as a fusion to small protein tags that facilitate purification, isolation and/or visualization. Preferably, peptides are expressed as a fusion to glu- tathione-S transferase, the affinity and specificity of binding to glutathione permitting purification using glutathione affinity resins, such as Glutathione Sepha- rose 4B beads (Amersham Biosciences), with subsequent elution with free glutathione. As another preferred alternatives, peptides can be expressed with a polyhistidine tag, a chitin-binding tag and seif-excising intein, a calmodulin- binding peptide tag, or a specifically excisable fragment of the biotin carboxylase carrier protein.

[0048] Peptides suitable for use in the present invention can also be cloned into expression vectors that provide transient expression in mammalian cells.

[0049] Host cell means any host comprising the expression vector and being capable of expressing the protein encoded by the vector. The host cell may be eukaryotic or prokaryotic. Possible host are bacteria, yeast, fungi and mammalian cells.

[0050] SAP30L-protein contains in a stretch of 49 residues (aa 29- 77 in human SAP30L) four cysteine and two histidine residues. These residues are completely conserved in a phylogenetic comparison of SAP30/SAP30L sequences from the fruitfly to human. According to the mass spectrometric analysis the N-terminal fragment comprising the novel peptide has a zinc- containing structure consisting of a C2CH module and a co-ordinated zinc ion (Table 1).

[0051] Many well-characterized DNA-binding elements contain a zinc finger motif. The peptides suitable for use in the invention have an intrinsic nucleotide binding activity, which is partly mediated through the zinc-containing structure. The intrinsic nucleotide binding activity refers to the ability of the peptide to bind to any DNA or RNA sequence including regulatory sequences, exons, introns or any non-coding sequence. In contrast to most of the known zinc-coordinated structures, the SAP30L peptide binds both DNA and RNA without any sequence specificity as shown in the L-EMSA experiments in Figures 1 and 5. The nucleic acid binding is dependent on an intact N-terminus that contains the C2CH-type zinc module, whose disruption abolishes the DNA binding. However, a peptide comprising amino acids 78-92 of SEQ ID NO: 1 or SEQ ID NO: 3 is able to bind DNA.

[0052] In one embodiment of the method for nucleic acid binding, a SAP30 or SAP30L peptide, or a functional fragment or a variant thereof, having an ability to bind nucleic acids and phopholipids is added to a nucleic acid containing solution. The peptide then forms a complex with the nucleic acids. When phospholipids or zinc chelating agents are added to the nucleic acid containing solution the nucleic acids are released from the peptide and can be recovered.

[0053] In another embodiment of the present nucleic acid binding method a fusion protein comprising a SAP30 or SAP30L peptide, or a functional fragment or a variant thereof, is formed and added to a nucleic acid con-

taining solution. The fusion protein forms then a complex with the nucleic acids. When phospholipids or zinc chelating agents are added to the nucleic acid containing solution the nucleic acids are released from the fusion protein and can be recovered.

[0054] Nucleic acid containing solution can be any solution that contains DNA or RNA in any format. It can be e.g. crude lysate of bacterial fermentation, or a PCR amplification reaction, or patient samples (e.g. blood and other fluids and tissues), or environmental samples (e.g. water system, crime scenes). Alternatively it can be cellular nucleotide sequence present within the cell. It is not necessary that the sequence is naturally occuring sequence of the cell. For example, a retroviral genome, which is integrated within hosts cellular DNA, would be considered a cellular nucleotide. The cellular nucleotide sequence can be DNA or RNA and includes both introns and exons. The cell and/or cellular nucleotide sequence can be prokaryotic or eukaryotic, including yeast, virus or plant nucleotide sequence.

[0055] The nucleic acids can be isolated from the solution with any conventional method, such as commercial DNA or RNA isolation kits or with traditional extraction with phenol-chloroform or equivaltent organic solvents, either manually or with special devices that are suitable for performing DNA or RNA isolation.

[0056] In one specific embodiment of the nucleic acid binding method the fusion protein-nucleic acid complex is bound to a solid phase, e.g. glutathione derivatized solid support, such as glutathione beads, and thus can be transferred from one container to another. The nucleic acids bound to the complex can be released by adding phospholipids. The fusion protein of the invention can bind the nucleic acid and the solid support simultaneously.

[0057] The method of the invention may be used in many applications where nucleic acids need to be transferred e.g. in DNA/RNA isolation, purification processes or in concentrating nucleic acid from large volume to small volume. It may also be used for detecting nucleic acis from solutions.

[0058] The SAP30/SAP30L peptides, or functional fragments or variants thereof, may be employed in variety of applications including diagnostics and as research tools. Advantageously, they may be used as diagnostic tools for identifying the presence of nucleic acid molecules in a complex mixtures e.g in patient samples.

[0059] The non-specific nucleic acid binding ability together with the specific phosholipid binding ability make the peptides disclosed herein a useful tool in zinc-finger based technologies. The peptide can be used by incorporating it to known zinc-finger structures, e.g. therapeutical zinc fingers. As one example, therapeutical zinc fingers have been used in regulation of IGF2 and H19 genes expression (loss of proper imprinting control at the IGF2- H19 domain is a hallmark of cancer and Beckwith-Wiedemann syndrome) (Jouvenot Y. et ai (2003) Gene Ther 10, 513-22). incorporation of the peptide to therapeutic zinc finger structures may increase the affinity of the zinc-fingers without disturbing their specificity. Moreover, the effect of the therapeutic zinc finger proteins is strengthened by increasing its affinity to the target promoter. The phospholipid binding ability and especially the function of the peptide as a lipo-switch provides a novel way of regulating the nucfeic acid binding.

[0060] The invention is illustrated by the following non-limiting examples. It should be understood, however, that the embodiments given in the description above and in the examples are for illustrative purposes only, and that various changes and modifications are possible within scope of the claims.

Examples

Example 1. Mass spectrometric detection of Zinc-dependent structure in SAP30L 1-92 peptide

[0061] cDNA encoding N-terminal 92 aminoacids of SAP30L gene was cloned into pGEX-4T1 (GST) vector (Amersham Biosciences). SAP30L 1- 92 GST-fusion protein was produced in Escherichia coli (BL-21 strain) and purified with Glutathione Sepharose 4B beads (Amersham Biosciences) according to the manufacturer's instructions. The fusion protein was eluted from the beads with reduced glutathione or cleaved from GST by prothrombin. The quantity and integrity of the GST-fusion proteins were checked using Coomassie-stained SDS-PAGE.

[0062] Mass spectrometric detection of Zinc-dependent structure in SAP30L 1-92 peptide was performed as follows. The prothrombin cleaved SAP30L 1-92 peptide was desalted on PD-10 columns (Amersham Biosciences Uppsala, Sweden), and concentration was estimated from the absorb- ance at 280 nm using <s≥ ₈ o = 2560 cnrf ¹ M ^"1 . Prior to measurements, the sample was further diluted with the appropriate solvents: CH ₃ CN/H ₂ O/HOAc (49.5:49.5:1.0 v/v, pH 3.2 for denaturing solution conditions, and 10 mM

NH ₄ OAc buffer (pH 6.8) for non-denaturing solution conditions. AN experiments were performed on a 4.7-T hybrid quadrupole Fourier-transform ion cyclotron resonance (Q-FT-ICR) instrument (APEX-Qe; Bruker Daitonics, Biilerica, MA, USA) ₁ interfaced to an external electrospray ionization (ESl) source (Apollo- II™). The samples were infused directly at a flow rate of 1.5 μl_ min ^~1 , with dry N ₂ serving as the drying (10 psi, 200 ⁰ C) and nebulizing gas. ESI-generated ions were externally accumulated in a hexapole ion trap for 0.5-1.0 s and transferred to an Infinity ICR ceil for Sidekick™ trapping, conventional "RF- chirp" excitation and broadband detection. A total of up to 256 co-added (1- Mword) time-domain transients were fast Fourier transformed prior to magnitude calculation and external frequency-to-m/z calibration with respect to the ions of an ES Tuning Mix (Agilent Technologies, Santa Clara, CA, USA). Al! data were acquired and processed with the use of Bruker XMASS 7.0.8 software. The results presented in Table 1 show the mass of 1-92 peptide in native (holo) and denatured (apo) form and the difference of these masses agrees well with one zinc atom.

Table 1.

[0063] The calculated and experimentally determined masses for wild type SAP30L 1-92.

peptide ⁸ m _exp (Pa) ⁰ m _ca ι _c (Da) ^b Am _eX p-caic (Da) Elemental composition ^d apo-w.t. 10413.33 10413.28 +0.05 C ₄₄ 7H72 ₆ N ₁₄₀ O ₁₃₇ S ₅

/ro/o-w.t. 10476.25 10476.20 +O05 ₄₄ 7H ₇₂₄ Ni ₄₀ Oi ₃₇ S ₅ Zn ₁

[0064] ^a The data is presented for the peptide comprising residues 1-94 (two extra amino acids from the vector).

[0065] ^b Experimentally determined, most abundant isotopic mass. [0066] ^c Calculated, most abundant isotopic mass based on the sequence- derived elemental composition. [0067] ^d In the case of zinc binding, a loss of two protons was considered.

Example 2. DNA binding

[0068] To confirm DNA binding and to explore the DNA-binding domains full-length GST-SAP30L and GST-SAP30 fusion proteins and a series of truncated GST-SAP30L-proteins were studied in a simplified EMSA-assay

which utilizes a commercial DNA-ladder (L-EMSA). 1 μg of the GST-fusion proteins prepared in a similar way as described in Example 1 were incubated with 0.5 μg of 1 kb DNA ladder (GeneRuier™, Fermentas) in PBS for 10 min at room temperature. The reactions were run on EtBr-containing 1% agarose gel with standard DNA gel loading buffer.

[0069] When full-length GST-SAP30L and GST-SAP30 fusion proteins were incubated with the DNA ladder, a marked shift in the mobility of the DNA was observed indicating DNA binding. The fusion proteins bind DNA fragments of all sized independently of their base sequences thus indicating non-sequence specific binding.

[0070] GST-SAP30L 1-92 protein was able to bind DNA, and therefore contains all determinants for interaction with DNA. Two polybasic regions were shown to be necessary for DNA binding. One of these is in the loop of the zinc-coordinating structure (aa 50-69), and the other region (aa 84-92) has been previously identified as a nuclear localization signal (NLS). A hydrophobic region (aa 78-84) between the zinc-coordinating structure and the NLS was also shown to be required for DNA binding, since a construct containing both the zinc-coordinating structure and the hydrophobic region (aa 1-84) was able to bind DNA whereas the zinc-coordinating structure alone (aa 1-77) was not. Similarly, a construct which includes both the NLS motif (aa 78-92) and the hydrophobic region is able to bind DNA but the NLS alone (aa 84-92) is not. The role of the NLS was further demonstrated by the full length GST-SAP30L- KAAAK construct, in which three basic residues in the phosphoinositide (Pl)- binding polybasic region have been mutated to alanines, thus having markedly reduced affinity for DNA as compared to wild type GST-SAP30L. The results are shown in Figure 1.

Example 3. Function of the Zinc-Switch

[0071] To explore whether DNA binding ability of SAP30L 1-92 is dependent on zinc, the GST-SAP30L 1-92 fusion protein was incubated with 1 ,10-o-phenanthroline, a zinc-cheiating agent, and an L-EMSA assay accoding to Example 2 was performed. As shown in Figure 2, 1 ,10~o-phenanthroline at 50 mM concentration abolished the DNA binding, as evidenced by a lack of shift in the mobility of DNA. The GST peptide alone, or the methanol solvent, elicited no mobility changes. The results thus show, that DNA is released from the GST-peptide-DNA complex to supernatant by adding zinc-chelating agent.

Example 4. Protein-Lipid Blot Assay

[0072] The interaction of purified fusion proteins with phosholipids was studied by two protein-lipid blot assays. GST-SAP30L, GST-SAP30, GST- SAP30L (1-92) and (78-92) fusion proteins were prepared in the same way as in Example 1. Protein-lipid blot assays were performed by adding 0.5 μg/ml of GST-fusion proteins on the lipid blot membrane. Lipid biot membrane {PIP strips) and arrays were purchased from Echelon Biosciences.

[0073] The relative affinities of SAP30L and SAP30 for various PIs were quantified by using their fusion proteins to probe a lipid blot that contained serial dilutions of eight different PIs (Figure 3A). The full-length GST- SAP30L bound most tightly to Pf(S)P, followed by PI(3)P and PI(4)P. PI(5)P- binding of GST-SAP30L was four-fold higher compared to PI(3)P, and eightfold higher compared to PI(4)P. GST-SAP30 bound to immobilized PIs in an identical manner, though with slightly lower affinities (Figure 3A).

[0074] The phopholipid-interaction of peptides GST-SAP30L (1-92) and GST-SAP30L (78-92) was studied using a lipid blot membrane containing 20- pmol spots of the following samples: lysophosphatidic acid (LPA), lysophospho- choJine (LPC), phosphatidylinositol (Ptdlns), Ptdlns(3)P, Ptdlns(4)P, Ptdlns(5)P, phosphatidylethanolamine (PE), phosphatidylcholine (PC), sphingosine 1 -phosphate (S1P), Ptdlns(3,4)P ₂ , Ptdlns(3,5)P _2! Ptdlns(4,5P) ₂ , Ptdlns(3,4,5)P ₃ , phos- phatidic acid (RA), phosphatidylserine (PS) ₁ and blank, according to the manufacturer's protocol (Echelon Biosciences) (Figure 3B). The membrane bound peptide was detected by anti-GST-Ab. Each protein-ϋpid biot experiment was repeated at least once. The peptides bound most strongly to phosphatidylinositol-3- phosphate (Ptdlns(3)P), Ptdlns(4)P and Ptd!ns(5)P (Figure 3). In addition, GST- SAP30L (1-92) bound also weakly to PI(3,4)P2, PI(3,5)P2, PI(4,5)P2, PI(3,4,5)P3 and PA.

Example 5. Function of the Lipo-Switch in nucleic acid binding

[0075] The function of the SAP30L 1-92-peptide as a lipo-switch was detected by an L-EMSA assay. The assay was performed as described in Example 2. The DNA ladder was incubated with 5 μmol of SAP30L 1-92- peptide, whereafter Ptdlns or Ptd!ns(5)P were added to the reaction in equivalent molar quantities (Figure 4). It was observed that an equivalent molar amount (5μM) of monophosphorylated Pl releases the DNA bound to peptide

almost completely and in 4 times excess DNA is released completely. Non- fosforylated lipid Ptdlns was not able to diminish binding.

[0076] In addition, the peptide was analysed by conventional EMSA for detecting peptide-RNA interaction. 5S ribosomal RNA probe was transcribed from the plasmid T7 promoter driven plasmid (Rosorius et al., 2000 J Biol Chem. 21 :275(16):12061-8) by using Riboprobe in vitro transcription system (Promega) according to the manufacturer's instructions. The probe was incubated with 0.5 μg of a GST-fusion protein on ice for 30 min in a buffer containing 10 mM Tris-HCI, pH 7.5, 100 mM KCI, 2 mM DTT and 4% glycerol, whereafter Ptdlns or Ptdins(5)P were added to the reaction in equivalent molar quantities (Figure 5). The reaction products were analyzed on a 6% non- denaturing polyacryiamide gel, dried and autoradiographed. As shown in Figure 5, a marked shift was detected in the mobility of the DNA after incubation with GST-SAP30L1-92 peptide, indicating a direct interaction with RNA. The mobility shift generated by binding of SAP30L1-92 peptide to RNA in the EMSA assay was diminished after addition of equivalent molar amounts of monophosphorylated PIs (PtdlnsδP), but not unphosphorylated PIs (Ptdlns).