KR20060135798A - Screening assays - Google Patents

Abstract

The present invention provides a method for modulating regulatory RNA- ligand binding, assays for identifying agents and oligonucleotides that change the thermodynamic probability of a secondary structure element of an RNA molecule.

Description

Screening method {SCREENING ASSAYS}

The present invention provides methods for modulating regulatory RNA-ligand interactions, for example for the identification, selection and identification of opener and closure nucleic acids for control manipulation of RNA secondary structures. In a preferred embodiment, the openers or closures of the invention are used to modulate RNA-ligand interactions and related downstream processes such as gene expression depending on the RNA secondary structure.

The present invention, for example, relates to the field of RNA biology, in particular RNA secondary structure analysis and prediction and RNA-ligand interactions dependent on RNA secondary structure. In particular, the present invention relates to RNA interactions with proteins involved in gene regulation, more particularly post-transcriptional regulation of gene expression, in particular control of mRNA stability, for example protein HuR (ELAVL1), to mRNA containing AU-rich elements. MRNA stabilization by binding. The invention further relates to, for example, the field of drug discovery, and more particularly to the identification and identification of pharmaceutical targets, the development of assays for high throughput screening and the profiling of compounds. Further the present invention controls the manipulation of RNA-ligand interactions related to diseases, in particular RNA-ligand interactions dependent on RNA secondary structural features, eg messenger RNA stability of genes related to diseases. RNA-ligand interactions, eg, manipulation of interactions between proteins HuR and AU-rich element mRNAs such as early response gene mRNAs, eg, mRNAs of TNFα or IL-2. The present invention includes the development of nucleic acid tools for the manipulation of gene expression, which relates to different but complementary approaches such as antisense techniques or RNAi.

Many regulatory processes involving RNA in cells depend critically on the formation of specific RNA secondary structures. In understanding the present invention, the secondary structure Ψ of the RNA molecule of sequence s is a list of base pairs (ie, pairs of nucleotides in the sequence) that satisfy the following conditions: ( i ) each nucleotide s _i is a maximum of one Participates in base pairs; ( ii ) each base pair ( s _i , s _j ) of Ψ is one of the canonical base pairs GC, CG, AU, UA, GU and UG; ( iii ) Base pairs satisfy non-pseudoknot conditions. That is, they are not crossed: two base pairs ( s _i , s _j ) and ( s _k , s _l ) of Ψ with i < j , k < l , and i < k mean j < k or j > l do.

Like other macromolecules, RNA molecules of the same sequence s can form many different structures represented by the set Σ (s). For each secondary structure Ψ in Σ (s), free energy by summing up the energy contributions for the stacked base pairs, hairpin loops, inner loops, bulges, and dodge loops F (Ψ) (free energy for the random coil structure) can be calculated by computer. This contribution was determined experimentally (Mathews DH et al., J. Mol. Biol. 1999, 288 (5): 911-40). The structure is not evenly distributed within the thermodynamic equilibrium ensemble of the structure, but the frequency of the structure depends on its stability (ie its free energy) and can be calculated by computer as follows.

는 RNA 분자 s의 분배 함수이고, T는 온도이며, R은 이상기체상수이다.In the formula,

Is the partition function of the RNA molecule s , T is the temperature, and R is the ideal gas constant.

As a result of the foregoing, secondary structural motifs or secondary structural elements required for a particular biological process are formed only by subset A ( s ) ⊆ Σ ( s ), not by all structures in the thermodynamic equilibrium ensemble of sequences. Can be. Structures forming the secondary structural motifs or elements required for a particular biological purpose are referred to throughout this invention as active or accessible structures herein. If a particular secondary structure motif can be formed only once in the secondary structure, the probability p _* of the accessible structure in the thermodynamic ensemble can be computed by summing up the probabilities of the individual structures in A ( s ).

Wherein Z _* refers to a constrained distribution function, that is, a distribution function of an ensemble of structures constrained to an accessible structure.

Computation of p _* is more complicated if the secondary structure motif can be formed multiple times in one structure. In this case, p _* is redefined with the probability of a structure forming one or more necessary motifs. The probability that the motif is formed at position B _i and the probability that the motif is formed at position B _j in the same structure are not independent. Therefore, the sum of the individual probabilities must be corrected for joint occurrence. For M site B _i ( i = 1 ... M ) in sequence s where a motif can be formed, p _* can be calculated using the exclusion-inclusion principle (eg, literature Meisner NC et al., Chembiochem 2004, 5 (10): 1432-47; see Hackermueller J. et al., Gene, 2005, published).

)은 앙상블 상에 구속이 없음 (따라서 p(

) = 1)을 의미하고, a = {B ₁ , B ₂ , ..., B _M }은 모티프가 모든 가능한 부위에서 형성되는 것을 의미한다. RNA 분자 M의 열역학은 짧은 올리고뉴클레오티드 O와 혼성화할 때 급격하게 변할 수 있다. 따라서, 올리고뉴클레오티드의 혼성화는 특정 2차 구조 모티프를 형성하는 RNA 분 자의 성향을 변하게 할 수 있다. 더욱 정확하게는, 하나 이상의 당해 구조 모티프를 형성하는 열역학적 앙상블 내의 구조의 확률은 RNA 분자 단독의 구조 앙상블과 비교했을 때 RNA-올리고뉴클레오티드 혼성물의 구조 앙상블에서 상이할 수 있다. 하나 이상의 당해 모티프를 형성하는 2차 구조의 전체적인 확률 p _*는 RNA 분자의 접근가능한 구조의 확률 p _* ^M , RNA-올리고 혼성물의 접근가능한 구조의 확률 p _* ^MO , 및 혼성화된 RNA와 혼성화되지 않은 RNA 간의 평형, 즉 올리고뉴클레오티드 [O] 및 RNA [M]의 평형 농도 및 혼성화 평형 상수 K _MO 로 표시되는 RNA와 올리고뉴클레오티드 간의 혼성화 에너지에 의존적이다. (i) 올리고뉴클레오티드가 RNA에 거의 상보적이고; (ii) 올리고뉴클레오티드와 RNA가 현저하게 자가 상보적이지 않고; (iii) 올리고뉴클레오티드가 과량으로 존재한다는 가정 하에, p_* ^MO에 의해 p_*의 근사값을 구할 수 있다(문헌 [Hackermueller J. et al., Gene, 2005, 출판중]).Where p ( a ) is the probability of the structure forming the motif at the subset a ⊆ { B ₁ , B ₂ , ..., B _M }, and the fraction of the distribution function p ( a ) = Z ( a Can be recalculated as / Z. Z ( a =

) Has no constraint on the ensemble (so p (

) = 1), and a = { B ₁ , B ₂ , ..., B _M } means that the motif is formed at all possible sites. The thermodynamics of RNA molecule M can change drastically when hybridizing with short oligonucleotide O. Thus, hybridization of oligonucleotides can alter the propensity of RNA molecules to form specific secondary structure motifs. More precisely, the probability of a structure in a thermodynamic ensemble that forms one or more of these structural motifs may be different in the structural ensemble of RNA-oligonucleotide hybrids as compared to the structural ensemble of RNA molecules alone. The overall probability p _* of the secondary structure forming one or more of these motifs is the probability p _* ^M of the accessible structure of the RNA molecule, the probability p _* ^MO of the accessible structure of the RNA-oligo hybrid, and not hybridized with the hybridized RNA. Equilibrium between RNAs, ie the equilibrium concentrations of oligonucleotides [ O ] and RNA [ M ] and the hybridization energy between RNA and oligonucleotides represented by the hybridization equilibrium constant K _MO . ( i ) the oligonucleotide is almost complementary to RNA; ( ii ) oligonucleotides and RNA are not significantly self-complementary; ( iii ) On the assumption that oligonucleotides are present in excess, an approximation of p _* can be obtained by p _* ^MO (Hackermueller J. et al., Gene, 2005, published).

Specific RNA-protein interactions are critically important in the regulation of numerous cellular mechanisms. Pre mRNA processing, nuclear (m) RNA export, RNA stability and degradation, other levels of regulation of gene expression, metabolic processes and viral life cycle regulation are critically dependent on the specific recognition of RNA molecules by protein factors . For many of these processes, RNA secondary structure motifs have been shown to enhance or enable recognition of (synonymous) sequence motifs, or "pure" RNA secondary structure motifs without sequence constraints have been recognized by regulatory proteins. . The apparent affinity of RNA protein interactions requiring specific RNA secondary structure motifs has recently been shown to directly depend on the probability of an accessible secondary structure (ie, forming a motif) within the thermodynamic equilibrium ensemble of RNA sequences:

는 겉보기 해리상수이고, K _d = [R _* ][P]/[RP]는 단백질과 접근가능한 RNA 분자 간의 친화력을 기술하는 미시적 해리 상수이다. [R], [R _* ], [P], [RP]는 RNA, 접근가능한 RNA, 단백질 및 RNA 단백질 복합체의 각각의 평형 농도를 나타낸다.In the formula,

Is the apparent dissociation constant and K _d = [ R _* ] [ P ] / [ RP ] is the micro dissociation constant describing the affinity between the protein and the accessible RNA molecule. [ R ], [ R _* ], [ P ], [ RP ] represent respective equilibrium concentrations of RNA, accessible RNA, protein and RNA protein complex.

Dependence on RNA secondary structure has recently emerged for the interaction between protein HuR and AU-rich urea mRNA. The AU-rich element (ARE) is a cis- acting element mainly present in the 3 'untranslated region (UTR) of the early response gene mRNA. These loosely defined elements are inherently characterized by a combination of AUUUA and UUAUUUA (U / A) (U / A) motifs and U-rich regions. Their presence targets messenger RNA for rapid degradation into cis action and their function is dependent on specific interactions with trans-acting factors. Of the 21 or more ARE-binding proteins identified thus far, the positive regulatory effect is present only in ubiquitously expressed HuR (ELAVL1, reference sequence accession no .: NP_001410) and its neuronal homologues HuB, HuC, HuD It was thought. The interaction between HuR and ARE-containing mRNAs leads to the stabilization of bound mRNAs and includes about 3000 genes, including many disease-related proteins and pharmaceutical targets in cancer, inflammatory, viral, allergic, vascular and infectious diseases. Expression can be determined (see, eg, Bakheet T. et al., Nucleic Acid Res. 2003, 60 (3): 499-511). A list of genes currently controlled by HuR is provided in Meisner NC et al., Chembiochem 2004, 5 (10): 1432-47, BMP6 (bone morphogenic protein 6), CCL11 (chemokine (CC) Motif) ligand 11), eotaxin, CSF2 (colony stimulating factor 2), GMCSF (granulocyte monocyte colony stimulating factor), FSHB (follicular stimulating hormone beta), IL1b (interleukin 1 beta), IL2 (interleukin 2), IL3 (Interleukin 3), IL4 (Interleukin 4), IL6 (Interleukin 6), IL8 (Interleukin 8), MYOD1 (Rogenic Factor 3), MYOG (myogenin), NF1 (Nuro Fibromine 1), PITX2 (paired-like homeodomain transcription factor 2), TNFα (tumor necrosis factor alpha), VEGF (vascular endothelial growth factor), CCNA2 (cyclin (cyclin) A), CCNB1 (cyclin B1), CCND1 (cyclin D1), CCND2 (cyclin D2), CD83, CDKN1A (cyclin-dependent kinase inhibitor 1A, ie p21 or Cip1), CDKN1B (Cyclin-dependent kinase inhibitor 1B, ie p27 or kip1), DEK, FOS (v-fos FBJ mouse osteosarcoma virus oncogene homolog, c-fos), HLF (liver leukemia factor), JUN (v-jun sarcoma virus 17 Oncogene homologue (bird), c-jun), MYC (v-myc avian leukemia virus oncogene homologue, c-myc), MYCN (v-myc avian leukemia virus-associated oncogene, neuroblastoma , n-myc), TP53 (tumor protein p53), HDAC2 (histone deacetylase 2), MMP9 (matrix metalloproteinase 9), NDUFB6 (NADH dehydrogenase (ubiquinone) 1 beta Subcomplex), NOS2A (nitric oxide synthase 2A), PLAU (urokinase plasminogen activator), PTGS2 (prostaglandin-endoperoxide synthase 2), COX2 (cyclooxygenase 2), SERPINB2 (serine (Or cysteine) proteinase inhibitors), PAI-2 (plasminogen activator inhibitor 2 ), UBE2N (ubiquitin-conjugation enzyme E2N), ADRB1 (beta-1-adrenergic receptor), ADRB2 (beta-2 adrenergic receptor), AR (androgen receptor), CALCR (calcitonin receptor), CDH2 (cadherin cadherin) 2, type 1), N-cadherin, GAP43 (growth-related protein 43), SLC2A1 (solute carrier family 2 member 1), GLUT1 (glucose carrier 1), PLAUR (urokinase plasminogen activator receptor ), SLC5A1 (solute carrier family 5), TNFSF5 (tumor necrosis factor (ligand) superfamily, member 5, CD154), ACTG1 (actin, gamma 1), CTNNB1 (catenin) (cadherin-associated protein) ), Beta 1), MARCKS (myristoylated alanine-rich protein kinase C substrate), MTA1 (transition related 1), PITX2 (paired-like homeodomain transcription factor 2), SLC7A1 (cationic amino acid transporter, CAT- 1) is included. Binding of the positive regulator HuR to ARE mRNA is determined by the presence of its binding site NNUUNNUUU within the single strand shape.

The present invention combines the aforementioned aspects, namely the ability to influence RNA thermodynamics by hybridization of short oligonucleotides, and the dependence of RNA protein interactions on the formation of certain secondary structural elements. The present invention provides a method of rationally designing short oligonucleotides that manipulate the probability of secondary structures forming particular secondary structural elements required for a particular biological process, such as secondary structural elements required for RNA-ligand interactions. do.

RNA secondary structure is of central importance in the regulation of many cellular processes that depend on RNA-ligand interactions such as translation, mRNA processing, metabolic pathways or other control mechanisms. The present invention provides methods for controlled manipulation of RNA secondary structure and related regulatory processes. Computer designed oligonucleotides hybridize to their target RNA molecules, thereby maximizing or minimizing the probability of certain secondary structural elements critical for the molecular interaction at hand. Such modulator (opener or closure) nucleic acids, for example, allow the regulatory hairpins to unfold, as well as destroy or assist any functional RNA structural element. Such artificially induced shape reconstruction may occur in regions near or far from the hybridization site in the target RNA sequence. This can be used to manipulate the associated regulatory process by hiding or presenting the recognition site of the regulatory factor. In particular, use is described for specifically controlling the expression of target genes involved in a disease at the level of HuR dependent mRNA stabilization. Given the disease relevance of many genes regulated by HuR, targeting mRNA stability by manipulating mRNA secondary structure by the methods of the present invention may be suitable as a novel platform strategy for therapeutic intervention. Thus, the method of the present invention (i) the development of specific target related assays and HT screens for identifying compounds that inhibit or enhance the structural changes induced by the adjustment factor for the disease related to a target mRNA, (ii) cell biology Or enhancing or silencing gene expression as a tool in an in vivo assay, ( iii ) identifying a drug target or ( iv ) profiling a compound, and ( v ) specifically "opening" an individual among multiple binding sites. Or means for studying the mechanism of biological function of a particular protein binding site by “closing”.

In one aspect, the present invention provides a method of modulating regulatory RNA-ligand interaction, comprising the following steps.

(a) defining and selecting secondary structural elements of RNA molecules required for recognition by ligands, eg proteins,

(b) calculating the thermodynamic probability of the secondary structural element of step (a) in the secondary structural ensemble of the RNA,

(c) calculating the thermodynamic probability of the secondary structural element of step (a) in the secondary structural ensemble of said RNA hybridized to at least partially reverse complementary oligonucleotides,

(d) determining oligonucleotides that change the thermodynamic probability of the secondary structural element beyond a defined range of probability thresholds,

(e) providing the oligonucleotide determined in step (d), and optionally

(f) hybridizing the RNA comprising the secondary structural element of step (a) to the oligonucleotide of step (e), and determining the effect of the hybridization on the thermodynamic probability of the secondary structural element.

Unless defined otherwise herein, the terms are defined as follows: Any RNA molecule with biological function, such as messenger RNA (mRNA), ribosomal RNA ( RNA from the group consisting of rRNA), small nuclear RNA (snRNA), transfer RNA (tRNA), micro RNA (miRNA), small nucleus RNA and spliciosome RNA. Preferably, the RNA is mRNA, such as AU-rich urea mRNA, for example BMP6 (bone morphogenic protein 6), CCL11 (chemokine (CC motif) ligand 11), eotaxin, CSF2 (colony stimulating factor 2), GMCSF (Granulocyte monocyte colony stimulating factor), FSHB (follicular stimulating hormone beta), IL-1b (interleukin 1 beta), IL-2 (interleukin 2), IL-3 (interleukin 3), IL-4 (interleukin 4), IL -6 (interleukin 6), IL-8 (interleukin 8), MYOD1 (round factor 3), MYOG (myogenin), NF1 (neupibromine 1), PITX2 (paired-like homeodomain transcription factor 2), TNFα (tumor necrosis factor alpha), VEGF (vascular endothelial growth factor), CCNA2 (cyclin A), CCNB1 (cyclin B1), CCND1 (cyclin D1), CCND2 (cyclin D2), CD83, CDKN1A (cyclin-dependent Kinase inhibitor 1A, ie p21 or Cip1), CDKN1B (cyclin-dependent kinase inhibitor 1 B, ie p27 or kip1), DEK, FOS (v-fos FBJ mouse osteosarcoma virus oncogene Fuselage, c-fos), HLF (liver leukemia factor), JUN (v-jun sarcoma virus 17 oncogene homologue (bird), c-jun), MYC (v-myc avian leukemia virus oncogene homolog, c -myc), MYCN (v-myc avian leukemia virus-associated oncogene, derived from neuroblastoma, n-myc), TP53 (tumor protein p53), HDAC2 (histone deacetylase 2), MMP9 (matrix metalloprotein) Inaze 9), NDUFB6 (NADH dehydrogenase (ubiquinone) 1 beta subcomplex), NOS2A (nitric oxide synthase 2A), PLAU (urokinase plasminogen activator), PTGS2 (prostaglandin-endoperoxide synthase 2 ), COX2 (cyclooxygenase 2), SERPINB2 (serine (or cysteine) proteinase inhibitor), PAI-2 (plasminogen activator inhibitor 2), UBE2N (ubiquitin-conjugation enzyme E2N), ADRB1 (beta -1-adrenergic receptor), ADRB2 (beta-2 adrenergic receptor), AR (androgen receptor), CALCR (calcitonin number Sieve), CDH2 (cadherin 2, type 1), N-cadherin, GAP43 (growth related protein 43), SLC2A1 (solute carrier family 2 member 1), GLUT1 (glucose carrier 1), PLAUR (urokinase plasminogen activity Factor receptor), SLC5A1 (solute carrier family 5), TNFSF5 (tumor necrosis factor (ligand) superfamily, member 5, CD154), ACTG1 (actin, gamma 1), CTNNB1 (catenin (cadherin-associated protein), beta 1 ), MARCKS (myristoylated alanine-rich protein kinase C substrate), MTA1 (transition related 1), PITX2 (paired-like homeodomain transcription factor 2) and SLC7A1 (cationic amino acid transporter, CAT-1) MRNA from the group of which is, for example, RNA is an mRNA such as IL-2 mRNA or TNF-α mRNA.

Ligands of the invention include, for example, RNA, DNA, peptides, proteins or small molecules such as proteins such as ARE binding proteins such as proteins of the ELAV family such as ELAVL1 (HuR), HuB, HuC, HuD, eg For example, molecules that bind to RNA molecules, including ELAVL1, are included.

Secondary structural motifs, used synonymously with the secondary structural elements of the present invention, include RNA secondary structural patterns consisting of paired and unpaired positions, e.g., eight loops that close a loop of five bases, such as hairpins. Hairpins of stacked bases are included. Alternatively, the secondary structural element or secondary structural motif may comprise a combined sequence / RNA secondary structural motif, such as a sequence pattern with secondary structural constraints, preferably a sequence pattern with a defined secondary structure, eg completely Sequence pattern NNUUNNUUU, wherein N is the IUPAC code for any RNA nucleotide, is single stranded. Preferably, secondary structural elements or secondary structural motifs are required for certain biological functions, e.g., binding of specific proteins to RNA or autocatalytic cleavage of molecules, preferably secondary structural motifs are characterized by It is necessary to bind to.

The probability of an RNA secondary structure within a thermodynamic equilibrium ensemble of a particular sequence forming a particular secondary structural element required for a biological process, such as the probability of an RNA secondary structure containing the sequence motif NNUUNNUUU in a completely single strand shape, is accessible herein. accessibility). The thermodynamic equilibrium ensemble can be any RNA molecule, such as an mRNA, or an ensemble of RNA hybridized to an oligonucleotide, such as mRNA hybridized to an opener or closure as defined below.

Oligonucleotides of the invention include all nucleotide oligomers of the defined sequence, preferably RNA, DNA, PNA (peptoid nucleic acid) or any 2'- or framework modifications such as 2'oxy-methyl or phosphorothio LNAs with locked-substituted (locked nucleic acids) are included. Oligonucleotides of the invention may also be used in labeled form, for example, at the 3 'or 5' end or at internal positions such as, for example, conventional labels such as biotin, cholesterol, digoxigenin, colloids, transition element complexes, carbohydrates, amino acids. It can be present as a substance, or as a fluorescent, radioactive, alkyl-, peptide or other tag or in the form labeled with a synthetic polymer such as polylysine or (poly) ethylene glycol. The length of the oligonucleotide depends on the secondary structure of the RNA and the desired sequence specificity, preferably the length of the oligonucleotide is 10 to 200 nucleotides, such as 10 to 100, preferably 10 to 50, such as about 20 nucleotides. Dog.

Oligonucleotides of the invention can act as openers or closures. The openers of the present invention have an accessibility of a hybrid of a particular RNA with the opener of a particular secondary structural element, eg, at least two times higher than a defined threshold compared to the accessibility of the RNA of the secondary structural element. Oligonucleotides are included that raise the threshold defined by value. The opener is partially complementary to the RNA, and is preferably exactly complementary to the RNA.

The closure of the present invention includes, for example, at least 0.5-fold lower accessibility of the hybrid of a particular RNA with the closure of a particular secondary structural element over a defined threshold compared to the accessibility of the RNA of the secondary structural element. Oligonucleotides are included that lower to a threshold defined by value. The closure is partially complementary to the RNA and is preferably exactly complementary to the RNA.

The invention further provides methods for identifying and selecting oligonucleotides that act as openers or closures for specific RNAs and specific secondary structural elements. The requirement is prior knowledge of the secondary structural elements required for the binding of the particular biological process in question, for example ligands, eg proteins. Secondary structural elements can be, for example, general knowledge or identified from the literature. For RNA-ligand interactions, the necessary secondary structural elements are described in detail in conventional methods, for example in Hackermueller J. et al., 2005, Gene (published), or in Meisner NC et. al. Chembiochem, 2004, 5 (10): 1432-47 can be identified using, for example, affinity data, using methods exemplified for the interaction between HuR and mRNA.

Accessibility p _* ^M is calculated for the RNA secondary structural element and for the RNA using, for example, the computerized methods provided in the computerized protocol. Depending on the computerized method, it may be possible to set the temperature at which accessibility is calculated. Preferably, the temperature is set to an ambient temperature at which the oligonucleotide is experimentally identified or applied, for example 37 ° for in vivo use.

Accessibility p _* ^MO of RNA-oligonucleotide hybrids is calculated for opener or closure candidates. The opener or closure candidate may be an arbitrarily selected oligonucleotide that is at least partially complementary to the RNA, or may be selected as a partially reverse complementary oligonucleotide that hybridizes to the RNA near where the secondary structural element may be formed. Can be. Preferably, p _* ^MO is assessed for a defined length, e.g., all oligonucleotides of 20 nucleotides, exactly complementary to the RNA.

If the difference between p _* ^M and p _* ^MO is beyond a certain threshold, for example, if p _* ^MO is twice as high as p _* ^M, the oligonucleotide is selected as an opener. If the difference between p _* ^M and p _* ^MO is beyond a certain threshold, for example if p _* ^MO is half of p _* ^M, the oligonucleotide is selected as a closure.

Optionally, such identified openers or closures can be hybridized to the RNA, for example ELISA (enzyme-linked immunosorbent assay), immunoprecipitation, filter binding assay, EMSA (= electrophoretic mobility shift assay), UV / VIS spectroscopy, NMR (nuclear magnetic resonance spectroscopy), fluorescence spectroscopy specifically focused on applications with single molecule sensitivity, such as Fluorescence Correlation Spectroscopy (FCS), Fluorescence Intensity Analysis (FIDA) Of hybridization to accessibility of secondary structural elements by known methods such as Distribution Analysis, or applications based on determination of Fluorescence Anisotropy or Fluorescence Resonance Energy Transfer (FRET). By determining the effect, it can be further confirmed experimentally.

In a preferred aspect, the invention provides a method of the invention wherein the RNA is IL-2 mRNA, the ligand is ELAVL1, and the oligonucleotide has a sequence selected from the group consisting of:

In another preferred aspect, the invention provides a method of the invention wherein the RNA is TNF-α mRNA, the ligand is ELAVL1, and the oligonucleotide has a sequence selected from the group consisting of:

In another aspect, the present invention provides the use of the method of the present invention to manipulate expression of a gene by altering the secondary structure of the corresponding RNA.

In a further aspect, the present invention provides oligonucleotides that change the thermodynamic probability of secondary structural elements beyond a defined probability threshold, as identified by the methods of the present invention.

In another aspect, the invention provides an oligonucleotide having a sequence selected from the group consisting of:

In a preferred aspect, the oligonucleotides identified by the method of the invention are from any chemical modification, for example from the group consisting of 2'-0-methyl-phosphothioate-, cholesterol-, biotin- and fluorescent dyes. RNA or DNA molecule or oligonucleotide of SEQ ID NO: 1 to 14, with the modification selected.

In another preferred aspect, the oligonucleotides identified by the methods of the present invention or oligonucleotides of SEQ ID NOs: 1-14 are PNA or LNA molecules.

Oligonucleotides identified by the methods of the invention or oligonucleotides of SEQ ID NO: 1 to SEQ ID NO: 14 are hereinafter referred to as "oligonucleotides of the present invention".

In another aspect, the invention provides for example the use of RNA secondary structures, for example to modulate RNA secondary structures such as RNA protein interactions, preferably mRNA protein interactions, and regulatory effects associated with such interactions. As a tool in the manipulations, the use of oligonucleotides selected from the group consisting of SEQ ID NOs: 1-14 or oligonucleotides identified by the methods of the invention for engineering regulatory RNA-ligand interactions is provided. In particular, oligonucleotides identified by the method of the present invention are controlled by gene expression, for example by AU-rich elements, preferably by manipulation of mRNA stability controlled by HuR illustrated in FIG. 1. Can be used to manipulate the expression of a gene.

The function of the opener or closure oligonucleotide provided by the present invention depends only on its ability to specifically hybridize with its target RNA. Thus, they are flexible in terms of chemical modifications. This allows to control the physical and biochemical properties of the opener or closure oligonucleotides. For example, single stranded RNA, DNA, PNA (peptoid nucleic acid) or LNA (locked nucleic acid) with any 2'- or framework modifications such as 2'oxy-methyl- or phosphorothioate-substituted can be used. Can be. In addition, the opener or closure oligonucleotides may be biotin, cholesterol, digoxigenin, colloid, transition element complexes, carbohydrates, amino acids, or at fluorescent, radioactive, alkyl-, peptide or other tags either at the 3 'or 5' end or at internal positions. Or with synthetic polymers such as polylysine or (poly) ethylene glycol. Openers or closure oligonucleotides can be used in in vitro, cellular or in vivo applications. For example, the opener or closure oligonucleotide of the present invention may be infected by a transfection method, such as by adjuvant mediated delivery, eg, by liposomes, DEAE dextran, calcium phosphate or other adjuvants. It can be delivered to cells by sex vectors, for example retroviral vectors, or by physical methods such as electroporation, microinjection or optoinjection. For in vivo assays, the opener or closure can be administered, for example, orally, by injection, eg, subcutaneously, intramuscularly or intravenously, by inhalation, rectally, topically, or microprojectile. Can be delivered by an approach ("Gene Gun").

For example, manipulation of gene expression by the opener or closure oligonucleotides of the invention may, for example, increase expression of disease related genes by the opener versus normal expression or closure or conventional methods of the invention, eg It may be applied to the identification of drug targets, for example by comparing phenotypic readings for reduced expression of the same gene mediated by RNAi, antisense or other knockdown methods. In another example, manipulation of gene expression by an opener or closure oligonucleotide of the invention can be achieved by, for example, expression of the gene increased by an opener versus normal expression or a closure or conventional method of the invention, eg RNAi. It can be used to study mechanisms in vivo, cell biological or biochemical assays by comparing phenotypic readings for reduced expression of the same gene mediated by antisense or other knockdown methods. The phenotypic readouts include RNA secondary structure, RNA tertiary structure, RNA-ligand complex level, RNA-ligand affinity, RNA oligomerization or multimerization, ligand oligomerization or multimerization, autocatalytic RNA cleavage, ligand morphology, RNA stubs Splicing, covalent RNA modification, RNA localization, RNA stability, RNA expression level, protein expression level, RNA or protein localization, cell proliferation, cell differentiation, cell migration, inflammation, tissue vascularization, tumor progression, angiogenesis Or a change in any other downstream effect of the RNA secondary structure manipulated by the oligonucleotides of the invention.

In another example, manipulation of gene expression by an opener or closure oligonucleotide of the invention can be achieved by, for example, expression of the gene increased by an opener versus normal expression or a closure or conventional method of the invention, eg RNAi. It may be applied to the profiling of agents, for example chemicals, by comparing the effect of the agents in reduced expression of the same gene mediated by antisense or other knockdown methods.

In another aspect, the present invention provides the use of an oligonucleotide selected from the group consisting of an oligonucleotide or SEQ ID NO: 1-SEQ ID NO: 14 identified by the method of the present invention to affect the stability of the RNA molecule.

In a further aspect, the present invention provides the following.

(I) hybridizing RNA comprising a secondary structural element necessary for recognition by a ligand to an oligonucleotide that changes the thermodynamic probability of the secondary structural element beyond a defined probability threshold in the presence and absence of a candidate compound ,

   (b) determining the effect of hybridization of the RNA to the oligonucleotide in the presence and absence of the candidate compound, and

   (c) identifying agents that modulate hybridization effects

A method for identifying an agent comprising a modulating effect of hybridization of an RNA molecule to an oligonucleotide.

(II) (a) hybridizing RNA comprising a secondary structural element necessary for recognition by a ligand to an oligonucleotide that changes the thermodynamic probability of the secondary structural element beyond a defined probability threshold,

    (b) hybridizing RNA comprising secondary structural elements necessary for recognition by the ligand to candidate compounds that are expected to have a similar effect to oligonucleotides,

    (c) determining the hybridization effect for steps (a) and (b), and

    (d) identifying an agent that mimics the hybridization effect of step (a)

An assay for identifying an agent that mimics the effect of hybridization of an RNA molecule to an oligonucleotide.

Candidate compounds include RNA fragments, DNA fragments, oligopeptides, polypeptides, proteins, antibodies, mimetic, small molecules such as low molecular weight compounds (LMW), preferably LMWs. Included are compounds (libraries) in which hybridization according to the invention can be expected.

The agent is one of the selected candidate compounds in which the hybridization effect as described above has been demonstrated.

Agents such as small molecules or other chemicals can be tested for inhibiting, enhancing or stimulating this effect, for example, by binding to specific shapes within the mRNA structure during rearrangement.

In a preferred aspect, the effect of hybridization in the assay of the invention is determined by measuring a signal related to the hybridization effect, the effect being determined by secondary RNA structure, tertiary RNA structure, RNA-ligand affinity, RNA oligomerization or multimerization, Consisting of ligand oligomerization or multimerization, changes in ligand shape, efficiency of downstream effects of RNA-ligand recognition, RNA splicing, covalent RNA modification, RNA localization, RNA stability, RNA translation, and changes in protein expression profiles Selected from the group.

Such assays can be realized by hybridizing oligonucleotides of the invention in the presence or absence of candidate compounds in the RNA, and by identifying phenotypic reads associated with the effects of the hybridization to identify agents that modulate or mimic the effects. The effect of the hybridization is for example determined by measuring the signal associated with the phenotypic readout as described above. For example, the assay can be used to allow genes controlled by the ARE to become drugs at the mRNA regulatory level. Assuming that cells use modulators such as small RNAs or proteins that induce mRNA structural changes that are strongly related to or similar to the openers or closures of the present invention, the openers or closures of the present invention may be used to design target specific mRNA stability assays. Can be used. Exemplary assay principles using the opener of the present invention are illustrated in FIGS. 11 and 12: The opener is structure grown for example detected by standard fluorescence spectroscopy methods such as fluorescence intensity, lifetime or fluorescence resonance energy transfer (FRET) measurements. Leads to heat.

As outlined in FIG. 13, expansion to cell assay formats is also possible. However, a requirement is to efficiently introduce probes and / or opener oligonucleotides into living cells. Once prototype assays are established, virtually all target mRNAs in the target platform of the 3,000 ARE gene will be screenable using conventional assay formats.

In a preferred aspect, the RNA in the assay is mRNA.

In another preferred aspect, the RNAs, ligands and oligonucleotides are as defined above.

In another aspect, the present invention provides the use of the assay of the present invention for high throughput screening.

In a further aspect, the present invention provides oligonucleotides selected from the group consisting of oligonucleotides or agents or SEQ ID NOs: 1-14 identified by the methods of the invention for use as pharmaceuticals.

In another aspect, the invention provides an agent identified by the assay of the invention or an oligonucleotide of the invention, and one or more pharmaceutical excipients such as fillers, binders, disintegrants, flow conditioners, lubricants, sugars and sweeteners. Provided are pharmaceutical compositions comprising suitable carriers and / or diluents, including, for example, fragrances, preservatives, stabilizers, wetting agents and / or emulsifiers, solubilizers, salts for adjusting the osmotic pressure and / or buffers.

In another aspect, the present invention provides a pharmaceutical composition of the present invention further comprising another pharmaceutically active agent.

Such compositions can be prepared according to conventional methods, for example by mixing, granulating, coating, dissolving or lyophilizing processes. The unit dosage form may contain, for example, about 0.5 mg to about 1000 mg, such as 1 mg to about 500 mg.

For use in pharmaceuticals, agents or oligonucleotides of the invention include combinations of one or more agents or oligonucleotides, for example agents or oligonucleotides.

Pharmaceutical compositions of the invention can be used for the treatment of disorders with etiology associated with downstream effects of RNA secondary structures engineered by such oligonucleotides or agents. For example, the downstream effect may be a substance, for example a protein encoded by a gene, for example a gene controlled by an AU-rich element, preferably a cytokine, chemokine, a growth factor, a prototype-oncogene, a virus. It may be the production of a protein selected from the group consisting of sex proteins, receptors, hormones or enzymes, preferably the substance is BMP6 (bone morphogenic protein 6), CCL11 (chemokine (CC motif) ligand 11), eotaxin , CSF2 (colony stimulating factor 2), GMCSF (granulocyte monocyte colony stimulating factor), FSHB (follicular stimulating hormone beta), IL-1b (interleukin 1 beta), IL2 (interleukin 2), IL-3 (interleukin 3), IL -4 (interleukin 4), IL-6 (interleukin 6), IL-8 (interleukin 8), MYOD1 (roundness factor 3), MYOG (myogenin), NF1 (neupibromine 1), PITX2 (pair De-like homeodomain transcription factor 2), TNFα (tumor necrosis factor alpha), VEGF (blood Vascular endothelial growth factor), CCNA2 (cyclin A), CCNB1 (cyclin B1), CCND1 (cyclin D1), CCND2 (cyclin D2), CD83, CDKN1A (cyclin-dependent kinase inhibitor 1A, ie p21 or Cip1), CDKN1B (cyclin -Dependent kinase inhibitor 1B, ie p27 or kip1), DEK, FOS (v-fos FBJ mouse osteosarcoma virus oncogene homologue, c-fos), HLF (liver leukemia factor), JUN (v-jun sarcoma virus 17 oncogene Homologues (birds), c-jun), MYC (v-myc avian leukemia virus oncogene, c-myc), MYCN (v-myc avian leukemia virus-associated oncogene, derived from neuroblastoma, n -myc), TP53 (tumor protein p53), HDAC2 (histone deacetylase 2), MMP9 (matrix metalloproteinase 9), NDUFB6 (NADH dehydrogenase (ubiquinone) 1 beta subcomplex), NOS2A (nitrogen oxide) Synthase 2A), PLAU (urokinase plasminogen activator), PTGS2 (prostaglandin-endoperoxide Synthetase 2), COX2 (cyclooxygenase 2), SERPINB2 (serine (or cysteine) proteinase inhibitor), PAI-2 (plasminogen activator inhibitor 2), UBE2N (ubiquitin-conjugation enzyme E2N), ADRB1 (beta-1-adrenergic receptor), ADRB2 (beta-2 adrenergic receptor), AR (androgen receptor), CALCR (calcitonin receptor), CDH2 (cadherin 2, type 1), N-cadherin, GAP43 (growth related Protein 43), SLC2A1 (solute carrier family 2 member 1), GLUT1 (glucose carrier 1), PLAUR (urokinase plasminogen activator receptor), SLC5A1 (solute carrier family 5), TNFSF5 (tumor necrosis factor (ligand) superfamily , Member 5, CD154), ACTG1 (actin, gamma 1), CTNNB1 (catenin (cadherin-associated protein), beta 1), MARCKS (myristoylated alanine-rich protein kinase C substrate), MTA1 (transition related 1) , PITX2 (paired-like homeodomain transcription factor 2) and SLC7A1 (cationic amino acid Carrier, CAT-1).

Treatment includes treatment and prevention.

For such treatment, appropriate dosages may, of course, depend, for example, on the chemical and pharmacokinetic data of the agents or oligonucleotides of the invention used, the individual host, the mode of administration and the nature and severity of the condition to be treated. Will change. In general, however, for satisfactory results in large mammals, such as humans, the recommended daily dosage ranges from about 0.01 g to about 1.0 g of the agent or oligonucleotide of the invention; For example, it is conveniently administered in divided doses up to four times a day.

Agents or oligonucleotides of the present invention may be administered by any conventional route, such as to the intestine including nasal, buccal, rectal, oral administration; Parenterally, including, for example, intravenous, intramuscular, subcutaneous administration; Or topically, including, for example, epicutaneous, intranasal, intratracheal administration; In the form of coated or uncoated tablets, capsules, for example ampoules, injection solutions or suspensions in the form of vials, creams, gels, pastes, inhaled powders, foams, tinctures, lipsticks, drops For example, in the form of a spray, or in the form of suppositories. Agents or oligonucleotides of the invention may be in the form of pharmaceutically acceptable salts, such as acid addition salts or metal salts; Or in glass form; Optionally in the form of solvates. The agents or oligonucleotides of the invention in salt form exhibit the same order of activity as the compounds of the invention in free form (optionally in solvate form).

Agents or oligonucleotides of the invention may be used alone or in combination with one or more other pharmaceutically active agents for the pharmaceutical treatment according to the invention. Formulations include fixed combinations in which two or more agents or oligonucleotides of the invention are present in the same formulation; Kits in which two or more agents or oligonucleotides of the invention in separate formulations are sold in the same package, eg, with instructions for coadministration; And free combinations where the agents or oligonucleotides of the invention are packaged separately, but instructions are provided for simultaneous or sequential administration.

1: Schematic diagram of the opener action

Without opener action, HuR binding is compromised by the inaccessibility of the binding site (denoted by 1). When the opener (designated 2) hybridizes to the target mRNA (mRNA α), the local secondary structure in the α mRNA is specifically tuned to present an accessible shape of the HuR binding site, resulting in any other HuR target mRNA ( For example, mRNA α is stabilized without affecting mRNA β).

Figure 2: Design of IL-2 Opener Oligonucleotides

HuR binding site accessibility at 37 ° p ^* _MO (i.e. fraction of accessible RNA) for 20mer oligonucleotides that are inversely complementary at a given hybridization site (starting position, x-axis) in the 3'UTR It is shown dependent on IL-2 3'UTR hybridization. IL-2 ARE is indicated by a white box and the NNUUNNUUU HuR binding site is represented by blocks in consecutive black lines at the opener start positions of about 50 and about 150. Significant increases in accessibility are limited to isolated "hotspots" near the HuR binding site. The positions of the experimentally tested opener molecules Op ₁ , Op ₂ , Op ₃ and Op ₄ are indicated and negative controls (N ₁ , N ₂ ) are also indicated (sequences detailed in Table 1). Openers Op ₁ and Op ₃ mainly target HuR binding sites in the ARE, with Op ₂ and Op ₄ directed to the second NNUUNNUUU motif.

Figure 3: IL-2 3 ' UTR Illustration of Opener Hybridization for

Minimum free energy (MFE) secondary structure of IL-2 3'UTR hybridized to IL-2 3'UTR (left panel) and opener Op ₁ (right panel, Op ₁ is indicated by dark lines). The NNUUNNUUU element is indicated by a small dot in the right panel. As illustrated in the MFE shape of the composite, the opener shifts the equilibrium into a shape with an accessible (ie single stranded) NNUUNNUUU element along the model depicted in FIG. 1. Importantly, since the ensemble of structures is related to opener prediction, the opener effect need not necessarily be reflected in the MFE structure. The MFE secondary structure for the 3'UTR-opener complex is computed using Cofold (available in Vienna RNA package).

Figure 4: IL -2 openers IL -2 3 ' UTR For In vitro HuR  Increase affinity

The apparent affinity of the recombinant HuR for IL-2 3'UTR is determined by 1D-FIDA detection in the presence and absence of an opener. All four IL-2 specific openers tested enhanced HuR association with IL-2 3'UTR, which is reflected by a decrease in apparent dissociation constant K _d ^app ( K _d ^app = 11.80 ± as Op ₁₎ . 1.48 nM; Op ₂ roneun ^{_{K d app = 18.91 ± 1.91 nM}} ; Op 3 roneun ^{_{K d app = 8.38 ± 1.18 nM}} ; Op 4 roneun ^{_{K d app = 19.52 ± 2.20 nM}} ; openers without ^{_{K d app = 32.77 ± 4.48 nM}} ; IL-2 3'UTR is 0.5 nM; openers are 25, 25, 5 and 1 nM, respectively. Oligonucleotide negative control for IL-2 3'UTR hybridization of the oligonucleotides should not interact with the HuR unaffected (N ₁ to the ^{_{K d app = 32.91 ± 6.34 nM}} ; N 2 roneun K _d ^app = 32.77 ± 3.72 nM; the concentration of N ₁ and N ₂ is 25 nM) (A). The affinity increase induced by opener hybridization shows a saturation curve with half maximum saturation at an opener concentration of 0.38 nM (apparent affinity of Op ₃ hybridization = 134 (± 54) pM. HuR for opened IL-2 3'UTR. The apparent affinity of the binding all approaches the maximum at K _d ^app of 8.57 ± 1.33 nM at 1.56 nM; IL-2 3'UTR is 0.5 nM in all experiments) (B). The presence of Op _1, an increase in the density of the opener to the minimum value of the K _d ^app = 11.80 ± 1.48 nM 1.56 nM in the Op ₁ concentration dissociation constant is reduced (IL-2 3'UTR was 2.5 nM in all experiments) . For Op ₁ , increasing the concentration beyond this optimum reverses the effect and increases the dissociation constant again.

Figure 5: IL -2 mRNA  Opener endogenous HuR - IL2 mRNA  Increase association

After treatment with or without opener or negative control oligonucleotides Op ₁ , Op ₂ , N ₂ and Op _T , HuR mRNA complexes are co-immunoprecipitated from lysates of human PBMCs. HuR bound IL-2 mRNA is quantified by real-time RT-PCR. The amount of IL-2 mRNA is normalized to the level (empty bar) in untreated cells. Openers are added at 2.5 μM (hatched bars) or 10 μM (solid bars) and negative controls N ₂ and Op _T are added at 10 μM. Both openers increase HuR mRNA complexation to 6.5-fold (Op ₁ ) or 3.1-fold (Op ₂ ) higher levels.

Figure 6: IL -2 mRNA  Opener IL-2 mRNA  Inhibits degradation

The degradation of endogenous IL-2 mRNA is monitored in human PBMC lysate. The addition of Mg ^{2 +} when (t = 0 min), the amount of remaining IL-2 mRNA in (A) 10 μM, (B ) 25 μM and (C) openers Op of 40 μM concentrations _1, Op ₂ or N ₂ Quantitation over time by quantitative real-time RT-PCR with and without. All data represent mean values from three or more independent samples and are normalized to the level at time point t = 0 min. Fit data to first order exponential decay (solid line: no opener; dashed line: N ₂ ). IL-2 mRNA has a _{half-life of} t _½ = 8.34 ± 0.96 min (A), t _½ = 8.44 ± 1.98 min (B), and t _½ = 8.84 ± 2.19 min (C) in the absence of an opener (white circle), As well as rapid degradation to (x) t _½ = 6.82 ± 1.96 min (A) in the presence of 10 μM negative control N ₂ . The addition of openers Op ₁ (black circles) or Op ₂ (black triangles) promotes transient IL-2 mRNA stabilization in a concentration dependent manner (the concentration of openers is 10 μM for (A), 25 μM for (B), (C) 40 μΜ). At concentrations of 40 μM (C), Op ₁ blocks degradation over a total incubation time of 70 minutes. Op ₂ targets another HuR binding site but shows a similar stabilizing effect (black triangles, (B) and (C)). EF-1α but not ARE mRNA remains stable over the entire observation time of 70 minutes (B).

Figure 7: IL -2 opener oligonucleotides IL -2 mRNA  Specifically promotes stabilization

The specificity of mRNA stabilization induced by openers is tested by monitoring their effect on the breakdown of other ARE containing cytokine mRNAs (TNF-α (A) and IL-1β (B)). TNF-α and IL-1β mRNA degradation is characterized by half lives of t _½ = 36.0 ± 2.2 minutes (white circle, (A)) and t _½ = 37.6 ± 5.6 minutes (white circle, (B)), respectively. In the presence of IL-2 specific openers Op ₁ (black circles) or Op ₂ (black triangles, all 25 μM), TNF-α or IL-1β mRNA disruption is not altered.

Figure 8: TNF -α opener and Closure  design

HuR binding site accessibility p ^* _MO at 37 ° (ie fraction of accessible RNA) is reversed to the reverse complementary 20-mer oligonucleotide at a given hybridization site (starting position, x-axis) within mRNA (reference sequence NM_000594) Dependent on TNF-α mRNA hybridization. TNF-α 3'UTR is drawn with black lines, ARE is shown with white boxes, and NNUUNNUUU HuR binding sites are shown with black small boxes. Hybridization sites with significant changes in accessibility are clustered in “hot spots” that are predominantly around the HuR binding site but also present far from the binding site. Notably, putative "closure" positions are also identified and are expected to significantly reduce NNUUNNUUU accessibility. Interestingly, these locations are located within the opener hot spot area. The positions of putative openers, closures and negative control oligos are also indicated by small boxes, respectively. The sequences of the identified TNF-α openers / closures (Op _A , Op _B , Cl _C , Op _E , Op _H ), including experimentally tested Op _T , Cl _T and N _T , are detailed in Table 1.

Figure 9: TNF-α openers increase in vitro HuR affinity for TNF-α 3'UTR.

The apparent affinity of recombinant HuR for TNF-α 3'UTR is determined by the 1D-FIDA assay in the presence and absence of openers, closures or negative control oligonucleotides. TNF-α closure Cl _T significantly reduces the association of HuR with TNF-α 3′UTR, which is reflected in the increase in the apparent dissociation constant K _d ^app . This effect is correlated with the concentration of the closure. The effect is close to the maximum with ~ 2.5-fold Kd increase at the highest concentration of the closure (Cl _T : K _d ^app = 13.80 ± 2.41 nM, without closure: K _d ^app = 5.63 ± 0.87 nM) (A). The opener Op _T increases HuR's affinity for TNF-α 3'UTR to a factor of ˜2 (B), which is in good agreement with the computerly predicted effect (FIG. 8). However, at opener concentrations above 0.5 nM, the effect reverts back to the increased dissociation constant. Hybridization with negative control oligonucleotide N _T does not affect HuR-TNF-α 3′UTR affinity over the entire concentration range of the experiment (C). TNF-α 3'UTR concentration is 1 nM in all experiments.

10: TNF-α openers specifically promote TNF-α mRNA stabilization

Openers designed for TNF-α (Op _T , see FIG. 8 and Table 1) specifically stabilize TNF-α mRNA (A) and do not affect IL-1 β mRNA levels (B). (Source: no opener; star: 25 μM opener Op _T ).

11: Opener based In vitro  Method Design I: Shape Switch switch )

로 표시됨)에 대한 진단이 된다. (A)에 묘사된 바와 같이, 컴퓨터로 설계된 오프너에 의해 유도된 HuR 결합 부위의 오프닝은 FRET 효율에서의 감소에 의해 검출된다. (B) 저분자량 화합물이 상이한 작용 방식에 의해 이러한 재배열을 방해할 수 있다. mRNA에 결합함으로써, 이들은 오프너 혼성화와 직접적으로 경쟁할 수 있거나, 또는 클로징된 형상으로 mRNA를 국소적으로 동결시킬 수 있다. 오프너의 존재 하에서의 지속적인 에너지 전달을 특징으로 하는 이같은 화합물은 HuR이 mRNA에 결합하는 것을 방지함으로써 상응하는 표적 유전자를 하향 조절시킬 것이다. 반면에, 오프너 효과는 오프닝된 형상을 안정화시키는 화합물에 의해 증강될 수 있다. 이러한 화합물뿐만 아니라 오프너에 의해 유도된 형상 스위치를 모방하는 것들은 오프너의 존재 또는 부재 하에 각각 감소된 에너지 전달에 의해 동정된다. HTS 포맷에 대한 개조를 위한 주요 이슈는 부위 특이적으로 이중 표지된 mRNA의 충분한 양으로의 제조일 것이다. 따라서 별법 전략이 도 12에 개요된다. Exemplary assay principles for the identification of compounds acting on ARE mRNA shape are illustrated. Shape rearrangement induced by the opener is detected, for example, by suitably placed fluorophores that make up the FRET pair. Strategically placed within the mRNA, their distance and thus the FRET efficiency opening or closing the HuR binding site (

Is indicated by. As depicted in (A), the opening of the HuR binding site induced by the computer designed opener is detected by a decrease in FRET efficiency. (B) Low molecular weight compounds may interfere with this rearrangement by different modes of action. By binding to mRNA, they can compete directly with opener hybridization or can locally freeze the mRNA in a closed shape. Such compounds, characterized by sustained energy transfer in the presence of an opener, will down regulate the corresponding target gene by preventing HuR from binding to mRNA. On the other hand, the opener effect can be enhanced by a compound that stabilizes the opened shape. Mimicking these compounds as well as the shape switches induced by the openers are identified by the reduced energy transfer, respectively, in the presence or absence of the openers. A major issue for modifications to the HTS format will be the preparation of sufficient amounts of site specific double labeled mRNA. An alternative strategy is thus outlined in FIG. 12.

12: Based on opener In vitro  Method design II : HuR  Combination

Alternatively, one can redirect the assay strategy to the detection of HuR binding events by measuring the energy transfer between the two fluorophores on the mRNA and HuR. mRNA labels can be (A) covalently attached to the 3 'end (as described, eg, in Qin PZ et al., Methods, 1999, 18 (1): 60-70) or (B) openers May be introduced via oligonucleotides. In this setup, the shape rearrangement induced by the opener is indirectly measured by the determination of the HuR mRNA association, which is a subsequent step. Thus, HuR inhibitors will produce the same signals as compounds that act on mRNA shape and need to be sorted on the appropriate counterscreen.

13: mRNA  Opener-based strategy for cell assays

로 표시됨)에 충분히 가깝게 혼성화되면, 에너지 전달 (즉 도너(donor) 켄칭(quenching)/어셉터(acceptor) 민감화)이 클로징된 형상과 오프닝된 (즉 HuR이 결합된) 형상을 구별하는 바람직한 검출 방식이다. 도 11의 도해와 유사하게, 오프너에 의해 유도되는 효과를 저해하거나 또는 증강시키는 화합물은 오프닝된 mRNA 형상 대 클로징된 mRNA 형상의 구별을 기초로 동정될 수 있다. 본질적으로 "오프닝된" mRNA를 사용하는 카운터스크린이 HuR 저해제를 분류하는데 적절할 수 있다.Exemplary strategies for cellular assay design for ARE mRNA shape switches are outlined (A). Target mRNAs are specifically visualized by appropriately designed fluorescent probes (eg Cy5). Probes are computerized to ensure minimal impact on thermodynamic mRNA ensembles and compete with structural rearrangements induced by openers. Endogenous HuR is labeled by fusion to fluorescent proteins (eg CFP). In the closed mRNA shape, two fluorophores (HuR, mRNA) will be spatially separated. Upon binding of HuR induced by the opener, two fluorescent molecules will be localized together on the mRNA, which can be detected, for example, by high resolution confocal imaging (B). Alternatively, mRNA specific labels are placed directly on the opener oligonucleotides. In this setup, only the opened mRNA molecule is detected and the mRNA structure ensemble is not affected by the (additional) probe. Probe is a HuR binding site (

Hybridization close enough to the desired detection scheme, where energy transfer (ie donor quenching / acceptor sensitization) distinguishes the closed and opened (ie HuR coupled) shapes to be. Similar to the diagram of FIG. 11, compounds that inhibit or enhance the effects induced by the opener can be identified based on the distinction of the opened mRNA shape to the closed mRNA shape. Counter screens using essentially "opened" mRNA may be suitable for classifying HuR inhibitors.

Legend for the table:

Table 1: Identified Openers / Closure  Oligonucleotide

The sequence of putative openers or negative control oligoribonucleotides identified for IL-2 or TNF-α is detailed. Openers / closures selected for experimental confirmation, as well as negative control oligos, are indicated in bold. The sequence is reverse complementary to the above-described region in the target mRNA and is provided in a 5 'to 3' direction.

Table 2: Primers Used for RT PCR

The sequences of primers used for RT-PCR quantification of EF-1-α as internal control mRNAs as well as target mRNAs of IL-2, TNF-α and IL-1β are detailed in the 5 'to 3' direction.

In the examples below, all temperatures are given in degrees Celsius (°) and are not corrected.

The following abbreviations are used:

ACN acetonitrile

BSA Bovine Serum Albumin

dsDNA double stranded DNA

EDTA N, N, N ', N'-ethylenediaminetetraacetic

EF-1α Elongation Factor-1α

FCS fetal bovine serum

FIDA Fluorescence Intensity Distribution Assay

(1D-FIDA = 1D or 2D-FIDA = 2D FIDA)

FCS fluorescence correlation spectroscopy

hPBMC human peripheral blood mononuclear cells

IPTG Isopropyl-β-D-thiogalactopyranoside

LC / EI-MS liquid chromatography // electrospray ionization-mass spectroscopy

OD optical density

ORN oligoribonucleotide

PBS phosphate buffered saline

PCR polymerase chain reaction

PMA Phorbol-12-myristate-13-acetate

RRM RNA Recognition Motif

rt room temperature

RP-HPLC Reversed Phase High Performance Liquid Chromatography

RT-PCR Reverse Transcription Polymerase Chain Reaction

SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

ss single strand

TEAAc Triethylammonium Acetate

TMR Carboxytetramethylhodamine

UTR untranslated region

Example A-Experimental Protocol:

a) Preparation of Fluorescently Labeled RNA. The 5 'amino-C6 modified RNA was subjected to 5'-O-dimethoxytrityl-2'O-triisopropyloxymethyl-protected β'-cyanoethyl- (N, N-diiso on 394A Applied Biosystems). Published procedures using propyl-) nucleotide phosphoramidite (Glen Research) (see, eg, Chaix C. et al., Nucleic Acids Symp. Ser. 1989, (21): 45-6); Scaringe SA et al., Nucleic Acids Res. 1990, 18 (18): 5433-41) and manufacturer's protocol. ORNs are cleaved from the support following standard protocols, purified by base-, phosphate- and 2'-deprotected and denatured polyacrylamide gel electrophoresis. RNA concentrations were determined from Burger-Lambert from UV-absorption at 260 nm using an accurate molar extinction coefficient at 260 nm determined according to Gray DM et al., Methods in Enzymology 1995, 246: 19-34. Calculate according to Bouguer-Lambert-Beer law All ORNs are> 99% pure according to analytical RP-HPLC analysis (VYDAC C ₁₈ column, 5 μm, 300 μs, 4.6 mm × 250 mm, 0-50% in 45 minutes in TEAAc (0.1 M, pH 7.0)) Gradient elution with CH ₃ CN, UV-detection at 260 nm). TMR (molecular probe) is attached to the 5'aminolinker by standard reaction of the primary amine with the succinimidylester-activated fluorophore to form a stable carboxamide. Unreacted dye is hydrolyzed by the addition of hydroxylamine-hydrochloride. Labeled RNA was separated from the free dye by gel filtration, purified by RP-HPLC from unlabeled RNA, the concentration was determined by UV absorption spectroscopy as described above, and for dye absorption at 260 nm. Correct.

3 ′ UTRs are prepared by run-off transcription from dsDNA templates with T7 RNA polymerase (T7 MEGASCRIPT In Vitro Transcription Kit, Ambion). IL-7 and TNF-α (IL-2: nt 707-1035, TNF-α: nt 872-1568, GenBank Accession Nos. NM_000589 and NM_000594, respectively) using primers comprising the 3'UTR, the T7 promoter Incorporate into PCR transcription templates during PCR amplification. In essence, as described in Qin PZ et al., Methods 1999, 18 (1): 60-70, the transcript is oxidized at the 3 'end with Na (m-) IO ₄ and hydrazide. To activated Cy3 (AP Biotech). The product is then purified by RP-HPLC as described for synthetic ORN, desalted and transferred into aqueous solution by gel filtration. 1: 1 label stoichiometry is controlled by determination of Cy3 and RNA concentrations by UV / VIS absorption spectroscopy corrected for dye absorbance at 260 nm.

Preparation of Recombinant Human HuR . The coding sequence for full length HuR (amino acids 1-326, Reference SEQ ID NO: NP_001410) is amplified from cDNA prepared from activated human T-lymphocytes. The product is cloned directly into the NdeI and SapI sites of vector pTXB1 (IMPACT ™ -CN System, New England Biolabs), allowing the intein-chitin binding domain tag and C-terminus to be fused without additional amino acid insertion. On induction with IPTG (1 mM, 6 hours at 28 °), the fusion protein is expressed in E. coli ER2566 (New England Biolabs). Bacterial cells were treated with Tris / Cl (tris (hydroxymethyl) aminomethane, 20 mM pH 8.0), NaCl (800 mM), EDTA (1 mM) and Pluronic F-127 (0.2% w / v, Is dissolved by a continuous freeze / thaw cycle in the buffer of the molecular probe). After DNA digestion, lysates are cleared by ultracentrifugation and fusion proteins are captured on chitin agarose beads (New England Biolabs). After extensive washing with lysis buffer, the recombinant protein is recovered by thiol-induced self splicing on a column of intain tack with 2-mercaptoethanesulfonic acid (sodium salt, 50 mM) for 12 hours at 4 °. (See, eg, Cantor EJ et al., Protein Expr. Purif. 2001, 22 (1): 135-40). All co-eluted intein tacks and uncleaved fusion proteins are removed from the eluate in a removable second affinity step. Protein was gel filtered (DG-10 column, Bio) into storage buffer (Na ₂ HPO ₄ / NaH ₂ PO ₄ (25 mM) pH 7.2, NaCl (800 mM), Pluronic F-127 (0.2% w / v)) -Rad) and shock-frozen in liquid nitrogen in small aliquots and stored at -80 °. Under these conditions, full-length HuR is soluble without the presence of an advanced aggregation state (analytical size exclusion chromatography) and characterizes the CD-spectrum for the RRM domain (Manival X. et al., Nucleic Acids Res. 2001, 29 ( 11): 2223-30). Protein is> 99% pure according to LC / EI-MS, RP-HPLC and SDS-PAGE assays. N-terminal sequencing indicates the exact N-terminus where Met ₁ was lost quantitatively. To clearly determine the concentration, purified HuR was lyophilized, dissolved in guanidinium hydrochloride (6 M), and the concentration was determined by UV-spectrometry (Gill SC et al., Anal. Biochem. 1989, 182 (2): 319-26). This solution is used as an external standard for the determination of HuR concentration by RP-HPLC quantification.

0.5 kHz), TMR로의 조정 측정에서 결정된 공초점 부피 파라메터 A0 및 A1 (일반적으로 A0

-0.4 및 A1

0.08), 단일 성분 피트(fit). 이방성 r은 평행 및 수 직 극성에 대한 분자 휘도로부터 수학식 V로부터 계산되고 (Lakowicz, 1999, 상기 참조), 10회의 연속적인 측정으로부터 평균화된다. 2D- FIDA - Anisotropy HuR - RNA binding assay. Fluorescently labeled RNA was thermally denatured in assay buffer (PBS, Pluronic-F-127 (0.1% w / v), MgCl ₂ (5 mM)) at 80 ° C. for 2 minutes and cooled to room temperature ( Refolding at -0.13 ° C / sec) and dilute to 0.5 nM, which ensures an average <1 fluorescent particle in the confocal volume in the described setup (Ecotec BA, 2001, 2D-FIDA Quick Guide , Hamburg). The exact concentration in each sample is determined based on the particle number and the size of the confocal volume derived from the parallel FCS assessment, as provided by the adjustment parameters for the point diffusion function (EVOTEC BioSystems, 2001). Fluorescently labeled RNA is titrated against increasing concentrations of recombinant HuR (at least 11 titration points). HuR-RNA samples are incubated for at least 15 minutes at room temperature before each measurement. HuR-RNA complex formation is monitored under true equilibrium conditions by determination of fluorescence anisotropy with 2D-FIDA. Measurements are performed in 96 well glass bottom microtiter plates (Whatman) on an EvotecOAI PickoScreen apparatus at ambient temperature (constant at 23.5 °). The device based on the Olympus inverted microscope IX70 is equipped with two fluorescence detectors, a polar beam splitter in the fluorescence emission path, and an additional linear polar filter in the excitation path. HeNe laser (λ = 543 nm, laser power = 495 Hz) is used for fluorescence excitation. The excitation laser light is blocked from the optical detection path by the interference barrier filter at OD = 5. A 0.5 nM solution of TMR in assay buffer is used for the adjustment of confocal pinholes (70 μm) and determination of the G-factor of the device (Lakowitcz JR Principles of Fluorescence Spectroscopy, 2 ed., New York, 1999, Plenum Pubisihers) . Ten FIDA measurements are performed with a 10 second measurement time and a 40 microsecond rest time for each well. Molecular luminance q is inferred from the 2D-FIDA raw data for each polar channel using the FIDA algorithm (Kask P. et al., Biophys. J. 2002, 78 (4): 1703-13). Fitting parameter: background intensity in both channels determined from separate measurements of buffer (typically

0.5 kHz), confocal volume parameters A0 and A1 (typically A0, determined in calibration measurements with TMR)

-0.4 and A1

0.08), single component fit. Anisotropy r is calculated from Equation V from molecular luminance for parallel and vertical polarity (Lakowicz, 1999, supra) and averaged from 10 consecutive measurements.

_Wherein q _II , q, are the molecular luminances in the parallel and vertical polarity channels, and G represents the G-factor of the device.

Anisotropic data can be derived from the average normal depending on the degree of 1: 1 complex formation derived from the law of mass action (Daly TJ et al., J. Mol. Biol. 1995, 253 (2): 243-58). ) Based on the exact algebraic solution of the coupling equation describing the state anisotropy signal r , infer the equilibrium dissociation constant K _d ^app (nonlinear least-squares regression, GraFit 5.0.3, Erichacus software, London):

In the formula, [ RNA ₀ ]: total concentration of RNA, [ HuR ₀ ]: total concentration of HuR, r _min : anisotropy of free RNA, r _max : anisotropy of RNA-HuR complex, r : predetermined HuR ₀ and RNA _O Mean anisotropy for steady-state equilibrium in concentrations; Q: quenching, for 2D-FIDA- anisotropy measurement method, q _tot = q + Q _II in _{2q ┴ = q tot (max)} / q tot (min); q _II . All data presented are mean values from three or more independent experiments.

1D-FIDA HuR mRNA Binding Assay. Labeled mRNA or 3′UTR was thermally modified in assay buffer (PBS, Pluronic-F-127 (0.1% w / v), MgCl ₂ (5 mM)) at 80 ° C. for 2 minutes and at room temperature. Refold by cooling (-0.13 ° C./sec). Opener, closure or negative control ORN (MWG Biotech, see Table 1 for sequences) is added at a final concentration between 0.5 and 100 nM. The final concentration of Cy3-labeled mRNA is 0.5 nM and the exact particle number is determined as described for the 2D-FIDA anisotropy assay.

Labeled mRNA is titrated against increasing concentrations of HuR in the presence and absence of openers or negative control ORNs. HuR-mRNA complex formation is monitored by 1D-FIDA by determination of the molecular brightness change of Cy3 induced by HuR binding to RNA. HeNe laser (λ = 543 nm, laser power = 495 Hz) is used for fluorescence excitation, and the optical setup is similar to the setup for 2D-FIDA anisotropy measurement, using only one detection channel, and the polar beam splitter is optical Not on path Molecular luminance q is inferred from the 1D-FIDA raw data using the FIDA algorithm and averaged from 20 consecutive measurements (10 seconds each). The molecular luminance data is fitted based on an equation similar to Equation VI, adapted for fluorescence intensity measurements:

Wherein: q _min : molecular brightness of free RNA, q _max : molecular brightness of RNA HuR complex, q : mean molecular brightness for steady-state equilibrium at a given HuR ₀ and RNA _O concentration. All data presented are mean values from three or more independent experiments.

Preparation and Stimulation of Cells . hPBMCs were isolated from heparinized blood by Ficoll-Hypaque centrifugation, washed with PBS containing BSA (15% w / v), heat inactivated FCS (10% v / v), L-glutamine (2 mM ), Resuspended at 2 × 10 ⁶ / ml in RPMI 1640 (Gibco / BRL) supplemented with streptomycin (100 μg / ml) and penicillin (100 μ / ml) and incubated in a 37 ° CO ₂ incubator. hPBMCs are stimulated with PMA (25 ng / ml, Sigma-Aldrich) and anti-CD3 mAb (1 μg / ml, Pharmingen) for 4 hours.

Coimmunoprecipitation of HuR-mRNA Complex. For each immunoprecipitation, 5 × 10 ⁶ unstimulated cells were washed with PBS / BSA and stored at 4 ° C. in storage buffer (100 μl, Tris / Cl (10 mM) pH 7.5, NaCl ( 10 mM), EDTA (10 mM), Protease Inhibitor (Complete Mini EDTA free Protease Inhibitor Cocktail, Roche; 3 tablets per 50 ml lysis buffer) and Nonidet-P-40 (0.5% v / v) . RNAsin (0.4 μ / ml, Promega) and Superasln (0.2 μ / ml, Ambion) are added to inhibit nonspecific RNA degradation. The lysate is centrifuged at 15,000 × g and 4 ° for 4 minutes to pellet the nuclei. The clarified lysate is incubated with anti-HuR mAb (5 μg / ml, 19F12, molecular probe) for 5 minutes in the presence and absence of opener or negative control ORN (2.5 or 10 M) at 4 °. After addition of biotinylated anti (mouse) IgG mAb (10 μg / ml, Amersham Pharmacia), the immunocomplex is captured on Streptavidin Sepharose beads (Amersham Pharmacia). The beads are thoroughly washed with lysis buffer. HuR and complexed mRNA are eluted under acidic conditions (glycine / HCl (50 mM, pH 2.5), NaCl (50 mM), 95 ° C. pre-warmed). The eluate is passed through a BioSpin gel filtration column (BioRad) pre-equilibrated with H ₂ O by centrifugation. Coprecipitated RNA is quantified by real-time RT-PCR.

mRNA decay. 5 × 10 ⁶ stimulated hPBMCs are dissolved in lysis buffer as described above (250 μL) with or without opener, closure or negative control ORN (10, 25 or 40 μM). mRNA degradation is initiated by a (net concentration of 5 mM free Mg ^{+ 2)} was added MgCl ₂ in a clean lysate. The degradation reaction proceeds at room temperature and after various time points between 2 to 70 minutes incubation (50 μl aliquots for each time point), by the addition of buffer containing EDTA and guanidinium isothiocyanate (Quiagen) Is stopped. RNA is isolated using the RNeasy Miniprep RNA Isolation Kit (Quiagen) according to the manufacturer's protocol and treated with DNAase I for removal of residual DNA.

Quantitative Real - Time RT - PCR . RNA is reverse transcribed into cDNA using TaqMan RT PCR reagents (Applied Biosystems) and random hexamers for priming according to the following standard protocol. Control reactions for genomic DNA contamination are performed without the addition of reverse transcriptase. Quantitative RT-PCR is performed on ABI7700 devices (Applied Biosystems) with SYBR Green detection. EF-1α is used as an endogenous control. Primers used for RT-PCR are detailed in Table 2. While in vitro transcribed IL-2 mRNA is used for the assay, the ΔΔCt method is used for relative quantification of mRNA levels (as described in Applied Biosystems 2000, for example). All presented data are mean values from at least five identical independent samples and represent two or more independent experiments with cells from independent donors.

Example B Computerized Protocol

Calculation of accessibility p _* ^M of RNA using constraint distribution function . The calculation of p _* ^M for a secondary structural element defined by a sequence pattern with secondary structural constraints, eg, NNUUNNUUU, which is completely single stranded, is described. p _* ^M can be calculated directly as described for p _* in Equation III. To avoid numerical errors, p ( a ) is not calculated as a fraction of the distribution function, but as the difference in the ensemble free energy, where p ( a ) = exp ( W - W _a ) / RT ) where W is is the ensemble free energy of the RNA sequence, W _a is any portion above the structure of the thermodynamic secondary structure ensemble free energy of the element is bound to the ensemble to form the particular secondary structure element in a a. Ensemble free energy can be calculated using standard RNA secondary structure prediction software (Zuker M. Curr. Opin. Struct. Biol. 2000, 10 (3): 303-10). RNA libraries distributed with the Vienna RNA package (Hofacker IL et al. Monatshefte Chemie 1994, 125: 167-88) were used, which are described in Mathews DH et al., J. Mol. Biol. 1999, 288 (5): 911-40].

n _* /n으로 구해진다. 근사값 n _* /n은 n의 증가와 함께 실제 p _* ^M 으로 수렴한다. Calculation of accessibility p _* ^M of RNA using secondary structure samples . Alternatively, an approximation of p _* ^M can be obtained by counting the occurrence n _* of accessible structures within a set of (secondary) RNA secondary structures of size n having a thermodynamic equilibrium distribution. Such a set of RNA secondary structures can be generated using stochastic backing (Tacker M. et al., Eur. Biophys. J. 1996, 25: 115-30; Ding Y. et al. Nucelic Acids Res. 2003, 31 (24): 7280-301], which runs on RNAsubopt , a program distributed with the Vienna RNA package (Hofacker IL 1994, supra). Then, the approximation of accessibility is p _* ^M

It is found by n _* / n . The approximation n _* / n converges to the real p _* ^M with increasing n .

Calculation of accessibility p _* ^MO for oligonucleotide-RNA hybrids using partition function . Thermodynamics of RNA-RNA hybridization is well understood (Dimitrov RA et al. Biophys. J. 2004, 87 (1): 215-26). However, RNA secondary structure predictions are not currently being carried out where all possible structures of hybrids are considered and appropriate to directly study the impact of hybridization on accessibility. Thus, an approximation of all secondary structures of an RNA oligonucleotide duplex is such a secondary structure of RNA where the target site of the oligonucleotide cannot form internal base pairs, ie the RNA nucleotides involved in hybridization are constrained to be single stranded. The inventors speculate that it can be obtained by As a result, p _* ^MO can be calculated as p _* ^M using Equation III with additional constraints t for single stranded nucleotides due to hybridization:

( A ∪ t ) means that both constraints need to be implemented simultaneously. Again, the calculation is performed using ensemble free energy to minimize numerical errors as described above for the calculation of p _* ^M.

n _* (t)/n(t)이다[식 중, n(t)는 구속된 세트 내의 2차 구조의 전체 숫자이다]. Calculation of accessibility p _* ^MO of RNA using secondary structure samples . Similar to the calculation of p _* ^M , quadratic structure samples can be used to approximate p _* ^MO . It is again speculated that the effect of hybridization is limited by preventing hybridized nucleotides in RNA from pairing with internal bases. The frequency n _* ( t ) of accessible structure is counted in the set of secondary structures constrained to be single stranded within the hybridization region. Similar to the notation used for the distribution function approach, the approximation of accessibility is p _* ^MO

n _* ( t ) / n ( t ), where n ( t ) is the total number of secondary structures in the constrained set.

Example 1: HuR end IL -2 mRNA Associating to and thereby IL -2 mRNA  Operation of stability

Openers, closures and negative control ORNs are constructed for IL-2 and TNF-α mRNA and confirmed experimentally in vitro and in human primary cell lysates.

Plots are provided in FIG. 2 showing the effect of hybridization of 20-mer ORNs on the fraction of computer-accessible RNA structures calculated for the design of openers and negative control ORNs for IL-2. Notably, a marked increase in accessibility is limited to a limited number of "hot spots" within the mRNA, which are located primarily outside of the HuR binding site. In most other locations, hybridization leaves the local ARE shape largely unaffected. From these hotspots, four potential opener ORNs as well as two negative control ORNs are selected for experimental characterization (Table 1). In the first step, the ss ORN is used.

The opener effect is first checked in vitro. As measured in the 1D-FIDA assay, HuR binds with significantly high affinity to IL-2 3′UTR (281 nt) in the presence of any opener (FIG. 4A). The increase in affinity induced by the opener correlates with the opener concentration (FIGS. 4B and C). In addition, for Op ₁ this effect reverts at concentrations above a certain threshold (1.6 nM at 2.5 nM IL-2 3′UTR) and the HuR IL-2 3′UTR affinity is again reduced (FIG. 4C). One possible explanation may be that when above a certain concentration, this particular sequence also hybridizes to a second site within the IL-2 3'UTR, leading to adverse shape rearrangements.

Negative controls are performed with two IL-2 3′UTR specific 20mers that do not affect accessibility p (ssNNUUNNUUU). As demonstrated in FIG. 4, both ORNs do not affect HuR-IL-2 3′UTR association.

To demonstrate that opener ORN also functions in more complex cellular environments, endogenous HuR-IL-2 mRNA association in the cell line is quantified. Cellular lysates of hPBMCs are treated with opener ORN. This experimental approach allows the transfer effect of the opener to be excluded. Defined opener concentrations are achieved, and cellular stress responses induced by opener transfection are excluded from the experiment. The IL-2 mRNA-HuR complex is co-precipitated with or without openers and HuR bound IL-2 mRNA is quantified by real time RT-PCR. Nonspecific immunoprecipitation is ruled out using control antibodies (goat IgG, data not shown). Both openers increase HuR-IL-2 mRNA association levels by 6.5-fold in a concentration dependent manner (FIG. 5). No increase in HuR bound IL-2 mRNA was observed with the negative control ORN N ₂ or TNF-α specific opener Op _T under the same conditions. To confirm the potential of openers to adjust mRNA levels, we test whether the induced increase in HuR complex formation also antagonizes rapid ARE dependent mRNA degradation. The opener Op _1, Op _2, as well as monitor, Mg ^{+ 2} dependent of IL-2 mRNA decay observed in the hPBMC lysate in the presence and absence of N _2. The amount of addition of Mg ^{2 +,} remained IL-2 mRNA, which is quantified over time by real-time RT-PCR. In the absence of any openers, endogenous IL-2 mRNA is rapidly degraded (t _½ = 8.34 ± 0.96 minutes) and mRNA, not the ARE gene (EF-1α), is stable over 70 minutes of observation time (FIG. 6) . The half-life observed is the values previously described (eg, Raghavan et al., Nucleic Acids Res. 2002, 30 (24): 5529-38); t _½ (IL-2 mRNA in human primary T-cells). ) = 17 ± 10 minutes), indicating that this degradation system is effectively similar to the in vivo situation. In the presence of the opener Op ₁ (c = 10 μM), this degradation is completely stopped over a period of 15 minutes (FIG. 6A). At this time, 79.9% of the untreated IL-2 mRNA is already degraded. Also in long incubations, the collapse is significantly slowed down. At a concentration of 40 μM (FIG. 6C), Op ₁ blocks degradation over a total incubation time of 70 minutes. Op ₂ targeting another HuR binding site shows a similar stabilizing effect (FIG. 6, Op ₁ and Op _{2 at} 25 μM (B) and 40 μM concentration (C)). The degradation kinetics of IL-2 mRNA is not affected by hybridization with the negative control ORN N ₂ (10 μM N ₂ ; t _½ = 6.82 ± 1.96 min). To ensure that the IL-2 mRNA stabilization induced by the opener is indeed a specific effect, the mRNA stability of other ARE containing HuR targets is monitored in the presence or absence of the IL-2 specific opener of the present invention. TNF-α or IL-1β mRNA disruption is not affected by IL-2 specific openers (FIGS. 7A and B).

Example  2: HuR end TNF -α mRNA Associating to and thereby TNF -α mRNA  Operation of stability

A plot showing the effect of hybridization of the 20-mer ORN on the fraction of computer-accessible RNA structures calculated for the design of openers and negative control ORNs for TNF-α is provided in FIG. 8. Again, significant changes in accessibility cluster into “hotspots” within the mRNA, located adjacent to the HuR binding site but also remote to the binding site. From this hotspot, one potential opener ORN as well as putative closure and negative control ORNs are selected for experimental characterization (Table 1). In the first step, we use the ss ORN. The opener effect is confirmed in vitro. As measured in the 1D-FIDA assay, closure Cl _T significantly reduced the HuR affinity for TNF-α 3'UTR (697 nt). This effect correlates with closure concentration, closes to maximum effect at maximum closure concentration (5 nM), and decreases affinity by ˜2.5-fold (FIG. 9A). In contrast, the affinity is increased by the opener Op _T to a coefficient of ˜2 (for Op _T of 0.25 nM, FIG. 9B). Both effects are quantitatively consistent with the predicted accessibility change (FIG. 8). However, at higher concentrations of Op _T , the effect reverts and the HuR-TNF-α 3'UTR affinity again decreases. One possible explanation may be that if above a certain concentration, this particular sequence also hybridizes to a second site within the TNF-α 3'UTR, leading to adverse shape rearrangements. Importantly, hybridization with negative control oligo N _T did not alter HuR-TNF-α 3′UTR affinity (FIG. 9C).

Finally, it is tested whether the induced increase in HuR complex formation also antagonizes the rapid mRNA degradation of ARE. TNF-α mRNA disruption in hPBMC lysate in the presence and absence of Op _T is monitored. Upon addition of Mg ²⁺ , the amount of remaining TNF-α mRNA is assessed by RT-PCR over time. As a control, the effect of Op _T on the breakdown of IL-1β as another ARE control cytokine mRNA is measured. In the absence of Op _T , endogenous TNF-α mRNA rapidly degrades (t _½ = 36.0 ± 2.2 min, FIG. 10). The half-life observed is the values previously described (e.g., Raghavan A. et al., Nucleic Acids Res. 2002, 30 (24): 5529-38), t _½ (TNF-α) in human primary T-cells. mRNA) = 25 ± 11 minutes), indicating that this degradation system is effectively similar to the in vivo situation. In the presence of 25 μM Op _T , digestion is stopped over a 40 minute incubation time. At this time, degradation of untreated TNF-α mRNA has already proceeded to 53.1%. Also in long incubations, the collapse is significantly slowed down. Importantly, mRNA disruption of IL-1β mRNA as another ARE containing HuR target is not affected by Op _T , demonstrating that the opener-induced TNF-α mRNA stabilization is indeed a specific effect.

                         SEQUENCE LISTING <110> Novartis AG   <120> screening assays <130> 33610 <160> 30 <170> PatentIn version 3.2 <210> 1 <211> 20 <212> DNA <213> Artificial <220> <223> oligonucleotide <400> 1 aaggcctgat atgttttaag 20 <210> 2 <211> 20 <212> DNA <213> Artificial <220> <223> oligonucleotide <400> 2 aatataaaat ttaaatattt 20 <210> 3 <211> 20 <212> DNA <213> Artificial <220> <223> oligonucleotide <400> 3 tagagcccct agggcttaca 20 <210> 4 <211> 20 <212> DNA <213> Artificial <220> <223> oligonucleotide <400> 4 tgaaaccatt ttagagcccc 20 <210> 5 <211> 20 <212> RNA <213> Artificial <220> <223> oligonucleotide <400> 5 aaggccugau auguuuuaag 20 <210> 6 <211> 20 <212> RNA <213> Artificial <220> <223> oligonucleotide <400> 6 aauauaaaau uuaaauauuu 20 <210> 7 <211> 20 <212> RNA <213> Artificial <220> <223> oligonucleotide <400> 7 uagagccccu agggcuuaca 20 <210> 8 <211> 20 <212> RNA <213> Artificial <220> <223> oligonucleotide <400> 8 ugaaaccauu uuagagcccc 20 <210> 9 <211> 20 <212> DNA <213> Artificial <220> <223> oligonucleotide <400> 9 tcggccagct ccacgtcccg 20 <210> 10 <211> 20 <212> DNA <213> Artificial <220> <223> oligonucleotide <400> 10 tctggtagga gacggcgatg 20 <210> 11 <211> 20 <212> DNA <213> Artificial <220> <223> oligonucleotide <400> 11 acggcgatgc ggctgatggt 20 <210> 12 <211> 20 <212> DNA <213> Artificial <220> <223> oligonucleotide <400> 12 ttctggaggc cccagtttga 20 <210> 13 <211> 20 <212> DNA <213> Artificial <220> <223> oligonucleotide <400> 13 attccagatg tcagggatca 20 <210> 14 <211> 20 <212> DNA <213> Artificial <220> <223> oligonucleotide <400> 14 atcacaagtg caaacataaa 20 <210> 15 <211> 20 <212> DNA <213> Artificial <220> <223> oligonucleotide <400> 15 aatataaaat ttaaatattt 20 <210> 16 <211> 20 <212> DNA <213> Artificial <220> <223> oligonucleotide <400> 16 tagagcccct agggcttaca 20 <210> 17 <211> 20 <212> DNA <213> Artificial <220> <223> oligonucleotide <400> 17 tgaaaccatt ttagagcccc 20 <210> 18 <211> 20 <212> DNA <213> Artificial <220> <223> oligonucleotide <400> 18 agtgggaagc acttaattac 20 <210> 19 <211> 20 <212> DNA <213> Artificial <220> <223> oligonucleotide <400> 19 cataataata aatattttgg 20 <210> 20 <211> 20 <212> DNA <213> Artificial <220> <223> oligonucleotide <400> 20 ctggctccat ggggagggct 20 <210> 21 <211> 20 <212> DNA <213> Artificial <220> <223> oligonucleotide <400> 21 tgaggtcttc tcaagtcctg 20 <210> 22 <211> 20 <212> DNA <213> Artificial <220> <223> oligonucleotide <400> 22 acggcgatgc ggctgatggt 20 <210> 23 <211> 25 <212> DNA <213> Artificial <220> <223> oligonucleotide <400> 23 tcaccaggat gctcacattt aagtt 25 <210> 24 <211> 29 <212> DNA <213> Artificial <220> <223> oligonucleotide <400> 24 ggagtttgag ttcttcttct agacactga 29 <210> 25 <211> 18 <212> DNA <213> Artificial <220> <223> oligonucleotide <400> 25 aggcggtgct tgttcctc 18 <210> 26 <211> 24 <212> DNA <213> Artificial <220> <223> oligonucleotide <400> 26 gttcgagaag atgatctgac tgcc 24 <210> 27 <211> 20 <212> DNA <213> Artificial <220> <223> oligonucleotide <400> 27 gtacctgagc tcgccagtga 20 <210> 28 <211> 21 <212> DNA <213> Artificial <220> <223> oligonucleotide <400> 28 tcggagattc gtagctggat g 21 <210> 29 <211> 27 <212> DNA <213> Artificial <220> <223> oligonucleotide <400> 29 tttgagacca gcaagtacta tgtgact 27 <210> 30 <211> 22 <212> DNA <213> Artificial <220> <223> oligonucleotide <400> 30 tcagcctgag atgtccctgt aa 22  

Claims (18)

(a) defining and selecting secondary structural elements of RNA molecules required for recognition by ligands, eg proteins, (b) calculating the thermodynamic probability of the secondary structural element of step (a) in the secondary structural ensemble of the RNA, (c) calculating the thermodynamic probability of the secondary structural element of step (a) in the secondary structural ensemble of said RNA hybridized to at least partially reverse complementary oligonucleotides, (d) determining oligonucleotides that change the thermodynamic probability of the secondary structural element beyond a defined range of probability thresholds, (e) providing the oligonucleotide determined in step (d), and optionally (f) hybridizing the RNA comprising said secondary structural element of step (a) to the oligonucleotide of step (e), and (g) determining the effect of the hybridization on the thermodynamic probability of the secondary structural element Comprising, regulatory RNA-ligand interactions. The method of claim 1, wherein the RNA is IL-2 mRNA, the ligand is ELAVL1, and the oligonucleotide has a sequence selected from the group consisting of:

The method of claim 1, wherein the RNA is TNF-αmRNA, the ligand is ELAVL1, and the oligonucleotide has a sequence selected from the group consisting of:

Use of the method of any one of claims 1 to 3 for altering the expression of a gene by altering the secondary structure of the corresponding RNA. (a) hybridizing RNA comprising secondary structural elements necessary for recognition by a ligand in the presence and absence of candidate compounds to oligonucleotides that change the thermodynamic probability of the secondary structural element beyond a defined probability threshold, (b) determining the effect of hybridization of the RNA to the oligonucleotide in the presence and absence of the candidate compound, (c) identifying agents that modulate hybridization effects A method for identifying an agent comprising a modulating effect of hybridization of an RNA molecule to an oligonucleotide. (a) hybridizing RNA comprising a secondary structural element necessary for recognition by a ligand to an oligonucleotide that changes the thermodynamic probability of the secondary structural element beyond a defined probability threshold, (b) hybridizing RNA comprising secondary structural elements necessary for recognition by the ligand to candidate compounds that are expected to have a similar effect to oligonucleotides, (c) determining the hybridization effect for steps (a) and (b), and (d) identifying an agent that mimics the hybridization effect of step (a) An assay for identifying an agent that mimics the effect of hybridization of an RNA molecule to an oligonucleotide. 7. The method of claim 5 or 6, wherein the effect of hybridization is determined by measuring a signal related to the hybridization effect, the effect being secondary RNA structure, tertiary RNA structure, RNA-ligand affinity, RNA oligomerization or multimerization, Consisting of ligand oligomerization or multimerization, changes in ligand shape, efficiency of downstream effects of RNA-ligand recognition, RNA splicing, covalent RNA modification, RNA localization, RNA stability, RNA translation, and changes in protein expression profiles Assay selected from the group. The assay of claim 5, wherein the RNA is mRNA. The assay of claim 5, wherein the RNA, ligand and oligonucleotide are as defined in claim 2. Use of the assay of any one of claims 5 to 9 for high throughput screening. An agent identified by the assay of any one of claims 5 to 9 for use as a pharmaceutical. An oligonucleotide that changes the thermodynamic probability of a secondary structural element beyond a defined probability threshold, as identified by the method of claim 1. The oligonucleotide of claim 12 having a sequence selected from the group consisting of:

The oligonucleotide or oligonucleotide of claim 13, wherein the oligonucleotide is an RNA or DNA molecule with any chemical modification. The oligonucleotide or oligonucleotide of claim 13, wherein the oligonucleotide is a peptoid nucleic acid or a locked nucleic acid molecule. Use of the oligonucleotide of any one of claims 12-15 for engineering regulatory RNA-ligand interactions. Use of an oligonucleotide of any one of claims 12 to 15 to affect the stability of an RNA molecule. A pharmaceutical composition comprising an agent identified by the assay of claim 5 or an oligonucleotide of any one of claims 12-15, and one or more pharmaceutical excipients.

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