Nucleic acid-controlled catalytic RNA for trigger-responsive regulation

Abstract

A composition is reported that includes a pair of a first nucleic acid and a second nucleic acid, the first nucleic acid including, in that order, a first portion of a stem nucleic acid sequence, a cleavage site, a first stem-loop nucleic acid sequence, the 5' and 3' portions of which form a duplex, a first portion of a catalytic core sequence, a second stem-loop nucleic acid sequence, the 5' and 3' portions of which form a duplex, a second portion of the catalytic core sequence, and a second portion of the stem nucleic acid sequence that is complementary to, and thus forms a duplex with, the first portion of the stem nucleic acid sequence, and the second nucleic acid is complementary to at least a portion of the first or second portion of the stem nucleic acid sequence, and upon binding of the second nucleic acid to the first nucleic acid, results in a conformational change in the first nucleic acid, the conformational change being at least one of dissociation of the first portion of the first stem sequence from the second portion of the stem sequence and hybridization of one of said portions with the second nucleic acid, or dissociation of Loop I and Loop II, resulting in inactivation of catalytic activity, or association of Loop I and Loop II, resulting in activation of catalytic activity.

Description

[Technical field]

[0001] The present invention belongs to the field of catalytic RNA. More precisely, a self-cleaving or bond-forming catalytic RNA, such as a ribozyme, is combined with a nucleic acid binding site and introduced into the 3'UTR of a coding nucleic acid to control the expression of the coding nucleic acid depending on the presence of a nucleic acid sequence that binds to the catalytic RNA. [Background technology]

[0002] 2. Background of the Invention Ribozymes are self-cleaving RNA molecules that can be engineered to allow trigger-responsive control of their self-cleavage activity. When incorporated into the 5' or 3' UTR of mammalian mRNAs, these trigger-responsive ribozymes have been used for gene regulation purposes. The challenge is to engineer trigger-responsive ribozymes that have low leakiness in the off state and high expression levels in the on state. The majority of small molecule-responsive ribozymes allow for 2-10 fold changes in gene expression in response to small molecules such as theophylline, tetracycline, and guanine.

[0003] Recently, a morpholino-controlled ribozyme has been reported that demonstrated a greater than 100-fold difference in gene expression in the on versus off state (Zhong, G., et al., Nat Biotechnol. 2020 38 (2020) 169-175) (Non-Patent Document 1).

[0004] Mudiyanselage et al. (Methods 161 (2019) 24-34) reviewed second generation fluorogenic RNA-based sensors. They outlined that aptamers can bind and activate the fluorescence of different classes of fluorophores, so that target detection at different wavelengths is now feasible. For example, Spinach, also known as an RNA mimic of green fluorescent protein, is a 98-nucleotide long RNA aptamer that can specifically bind to the chromophore 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) and switch on the fluorescence.

[0005] Felletti and Hartig (Wiley Interdisciplinary Reviews: RNA, 8 (2017) 2-e1395) reviewed ligand-dependent ribozymes. They summarized the occurrence of ligand-dependent ribozymes in nature and the many examples realized by researchers who engineered ligand-dependent catalytic RNA motifs and methods to obtain ligand dependence.

[0006] However, most of the currently known and applied ribozyme switches have low dynamic range, high leakiness and / or suboptimal triggering properties and are not suitable for human gene therapy due to, e.g., unfavorable safety, toxicity or biodistribution properties.

[0007] The generation of targeted integration Chinese hamster ovary hosts using a Cre / Lox recombinase-mediated cassette exchange system to directly target one or two vectors simultaneously to a single locus has been reported by Ng et al. (Biotechnol. Prog. 37 (2021) 1-10) (Non-Patent Document 4).

[0008] A reversible RNA on-switch that controls gene expression of AAV-delivered therapeutics in vivo has been reported by Zhong Guocai (Nat. Biotechnol. 38 (2019) 169-175) (Non-Patent Document 5).

[0009] A general design strategy for protein-responsive riboswitches in mammalian cells and the engineering of a ribozyme cleavage-induced split fluorescent aptamer complementation assay have been reported by Simon Auslaender et al. (Nat. Meth. 11 (2014) 1154-1160; Nucl. Acids Res. 44 (2016) 1-7) (Non-Patent Document 6).

[0010] WO 2021 / 076563 (Patent Document 1) discloses RNA switches based on modified hammerhead ribozymes with improved activity, in which the most efficient regulator is an octaguanidine dendrimer-linked morpholino bond to an internal portion of the RNA switch downstream of the cleavage site. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] International Publication No. 2021 / 076563 [Non-patent literature]

[0012] [Non-Patent Document 1] Zhong, G., et al., Nat Biotechnol.2020 38(2020)169-175 [Non-Patent Document 2] Mudiyanselage et al. (Methods 161(2019)24-34) [Non-Patent Document 3] Felletti and Hartig(Wiley Interdisciplinary Reviews:RNA,8(2017)2-e1395) [Non-Patent Document 4] Ng et al.(Biotechnol.Prog.37(2021)1-10) [Non-Patent Document 5] Zhong Guocai(Nat.Biotechnol.38(2019)169-175) [Non-Patent Document 6] Simon Auslaender et al.(Nat.Meth.11(2014)1154-1160;Nucl.Acids Res.44(2016)1-7) Summary of the Invention

[0013] The present invention is based, at least in part, on the discovery that placing a self-cleaving or bond-forming catalytic RNA, e.g. a ribozyme, in combination with a nucleic acid binding site, e.g. in the stem region of the catalytic RNA, in the 3'UTR of an encoding nucleic acid, e.g. a (selection) marker gene such as eGFP, but before a polyA signal sequence, can be used to control expression of the encoding nucleic acid depending on the presence of a nucleic acid sequence that binds to the nucleic acid sequence binding site of the catalytic RNA.

[0014] The nucleic acids according to the invention allow different applications.

[0015] In one aspect of the invention, the nucleic acids according to the invention are used for selective activation or inactivation of expression of an operably linked encoding nucleic acid, which in one embodiment is selected from the group comprising nucleic acids encoding selectable markers, nucleic acids encoding non-therapeutic proteins, therapeutic nucleic acids and nucleic acids encoding therapeutic proteins.

[0016] In one aspect of the invention, the nucleic acids according to the invention are used for screening and selection of ribozyme-nucleic acid sequence pairs, where the nucleic acid sequence activates or inhibits the catalytic activity of the ribozyme.

[0017] Thus, one aspect according to the invention is a method for selecting ribozymes based on the leakiness or tightness of the regulatory properties of the regulatory nucleic acid.

[0018] Thus, one aspect of the invention is a composition comprising a first or second nucleic acid, or a pair of a first and a second nucleic acid, comprising: The first nucleic acid comprises the following sequence (5' to 3' or 3' to 5'): a first portion of the stem nucleic acid sequence, -cleavage site, a first stem-loop nucleic acid sequence (loop I), the 5' and 3' portions of which can / do form a duplex, - a first portion of a catalytic core sequence, a second stem-loop nucleic acid sequence (loop II), the 5'- and 3'-portions of which can / do form a duplex, a second portion of the catalytic core sequence, and a second portion of the stem nucleic acid sequence that is complementary to (and therefore capable of / will form a duplex with) the first portion of the stem nucleic acid sequence, the second nucleic acid is complementary to at least a portion of the first portion or the second portion of the stem nucleic acid sequence; Binding of the second nucleic acid to the first nucleic acid results in a conformational change in the first nucleic acid, said conformational change being at least one of the following: - dissociation of a first portion of the first stem sequence from a second portion of the stem sequence and hybridization of one of said portions with a second nucleic acid; - dissociation of loops I and II, which leads to inactivation of catalytic activity, - Association of loops I and II leads to activation of catalytic activity It is.

[0019] In certain embodiments of all aspects and embodiments of the invention, the first nucleic acid is a hammerhead ribozyme and the stem is loop III of the hammerhead ribozyme.

[0020] In certain embodiments of all aspects and embodiments of the present invention, the second nucleic acid is an LNA (locked nucleic acid).

[0021] A further aspect of the invention is a fusion nucleic acid comprising: - an encoding nucleic acid encoding a selectable marker or a therapeutic protein or a regulatory compound, in one preferred embodiment a fluorescent protein or an antibody or a regulatory protein, a nucleic acid comprising a first nucleic acid according to the invention, and - a polyadenylation signal sequence operably linked to the encoding nucleic acid (if the second nucleic acid sequence is catalytically inactive).

[0022] A further aspect of the invention is a mammalian cell comprising the first nucleic acid according to the invention or the fused nucleic acid according to the invention.

[0023] A further aspect of the invention is a method for selecting a pair of a first and a second nucleic acid according to the invention, comprising the steps of: - providing a library of fusion nucleic acids according to the invention, in which all or part of the stem nucleic acid in the first nucleic acid according to the invention is randomized (e.g. by error-prone PCR or synthesis or mutation, etc.), - Integrating members of the fusion nucleic acid library into a mammalian cell by targeted integration (using targeted integration, only a single member of the library is introduced into the genome of a cell, even if multiple members are provided / present); - depositing the cells once after integration to generate a single cell library, each member of the single cell library comprising one member of the fusion nucleic acid library; - determining members of the single-cell library that are capable of altering the catalytic activity of the first nucleic acid by the addition of a second nucleic acid (e.g., a second nucleic acid library) by determining the expression level of the encoding nucleic acid; - Selecting members of the single cell library in which the difference in the expression level of the encoding nucleic acid in the absence and presence of the second nucleic acid is greater than in other cells of the single cell library.

[0024] In certain embodiments of all aspects and embodiments of the invention, targeted integration is by recombinase-mediated cassette exchange (RMCE).

[0025] In certain embodiments of all aspects and embodiments of the invention, targeted integration is by double recombinase-mediated cassette exchange (double RMCE).

[0026] The use of double RMCE allows targeted integration of a fusion nucleic acid according to the invention and an additional nucleic acid, including a (second, different) selectable marker. By using two different integrated coding nucleic acids, for example, the expression level of the (second, further) selectable marker can be used for normalization of the expression level of the fusion nucleic acid (ensuring comparability of results; normalization) and for deselection of cells with incorrectly integrated fusion nucleic acid.

[0027] In certain embodiments of all of the aspects and embodiments of the invention, the elements of the first nucleic acid are listed in the 5' to 3' direction.

[0028] In a particular embodiment of all aspects and embodiments of the invention, an aspect is a pair of a first nucleic acid according to the invention and a second nucleic acid according to the invention.

[0029] In certain embodiments of all aspects and embodiments of the invention, the first nucleic acid is in the 3'-UTR of the coding sequence.

[0030] In certain embodiments of all of the aspects and embodiments of the invention, the first nucleic acid is after the coding sequence and before the polyA signal sequence.

[0031] In certain embodiments of all of the aspects and embodiments of the present invention, the coding sequence is in an expression cassette.

[0032] In certain embodiments of all aspects and embodiments of the invention, the coding sequence is 5' to the first nucleic acid and the polyA signal sequence is 3' to the first nucleic acid.

[0033] In certain embodiments of all of the aspects and embodiments of the present invention, the coding sequence and the polyA signal sequence are operably linked.

[0034] In certain embodiments of all aspects and embodiments of the invention, the second nucleic acid comprises a first portion that is complementary to at least a portion of the first portion or the second portion of the stem nucleic acid sequence, and a second portion that is complementary to a nucleic acid sequence that is 5' to the first portion of the stem nucleic acid sequence if the second nucleic acid is complementary to at least a portion of the first portion of the stem nucleic acid sequence, or a second portion that is complementary to a nucleic acid sequence that is 3' to the second portion of the stem nucleic acid sequence if the second nucleic acid is complementary to at least a portion of the second portion of the stem nucleic acid sequence.

[0035] In one preferred embodiment of all aspects and embodiments of the invention, the second nucleic acid is complementary to a second portion of the stem nucleic acid sequence.

[0036] In certain embodiments of all aspects and embodiments of the invention, the second nucleic acid comprises or consists of a first portion that is complementary to at least a portion of the stem nucleic acid sequence and a second portion that is not complementary to the first nucleic acid sequence.

[0037] In certain embodiments of all of the aspects and embodiments of the invention, the second nucleic acid is not complementary to the first nucleic acid outside of the stem nucleic acid sequence.

[0038] In one preferred embodiment of all aspects and embodiments of the present invention, the second nucleic acid is an LNA.

[0039] In certain embodiments of all aspects and embodiments of the invention, the second nucleic acid has a length of 8 to 21 nucleotides. In certain embodiments of all aspects and embodiments of the invention, the second nucleic acid has a length of 9 to 20 nucleotides. In a preferred embodiment of all aspects and embodiments of the invention, the second nucleic acid has a length of 11 to 18 nucleotides.

[0040] In certain embodiments of all aspects and embodiments of the invention, the second nucleic acid has a first portion of 8-13 nucleotides that is complementary to the stem nucleic acid sequence of the first nucleic acid and a second portion of 13-8 nucleotides that is complementary to a nucleic acid sequence 5' or 3' of the stem nucleic acid sequence of the first nucleic acid, i.e., not part of the first nucleic acid but outside the first nucleic acid. In one preferred embodiment of all aspects and embodiments of the invention, the second nucleic acid has a first portion of about 11 nucleotides and a second portion of about 7 nucleotides.

[0041] In certain embodiments of all of the aspects and embodiments of the invention, all of the nucleotides of the second nucleic acid have a phosphorothioate backbone.

[0042] In certain embodiments of all aspects and embodiments of the invention, the first nucleic acid comprises: as the first part of the stem nucleic acid sequence, the sequence CCU CGU (SEQ ID NO: 01), as cleavage site, the sequence CC (SEQ ID NO: 02), - the sequence of GGG GCU GGA CCG CCC C (SEQ ID NO: 03) as a first stem-loop nucleic acid sequence whose 5' and 3' portions form a double strand; - as the first part of the catalytic core sequence, the sequence GCU GAC GA (SEQ ID NO: 04), - the second stem-loop nucleic acid sequence, the 5' and 3' portions of which form a duplex, has the sequence GGC CCG CGG AGG GCC (SEQ ID NO: 05); - as the second part of the catalytic core sequence, the sequence GAA (SEQ ID NO: 06), - the sequence ACG AGG AGG (SEQ ID NO: 08) as a second portion of the stem nucleic acid sequence, which is complementary to and forms a duplex with the first portion of the stem nucleic acid sequence.

[0043] In certain embodiments of all aspects and embodiments of the invention, a first portion of the stem nucleic acid sequence has the sequence CTG AGG GTA GT (SEQ ID NO: 09) and a second portion of the stem nucleic acid sequence has the sequence ACT ACC CTC AG (SEQ ID NO: 10).

[0044] In certain embodiments of all aspects and embodiments of the invention, the second nucleic acid has the sequence ATT GTG CCT GAG GGT AGT (SEQ ID NO: 13).

[0045] In certain embodiments of all aspects and embodiments of the invention, the second nucleic acid comprises more nucleotides as nucleotides that are complementary to the first portion or the second portion of the first nucleic acid but not to other portions of the first nucleic acid.

[0046] In certain embodiments of all aspects and embodiments of the invention, the first nucleic acid is within the 3'-UTR of the expression cassette between the coding sequence and the polyA signal sequence.

[0047] In addition to the various aspects and embodiments depicted and claimed, the subject matter of the present disclosure is also directed to other aspects and embodiments having other combinations of features disclosed and claimed herein. Thus, the specific features presented herein, particularly as aspects or embodiments, may be combined with each other in other ways within the scope of the subject matter of the present disclosure, such that the subject matter of the present disclosure includes any suitable combination of features disclosed herein. The foregoing description of specific embodiments of the subject matter of the present disclosure is presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the subject matter of the present disclosure to the disclosed embodiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] Detailed Description of the Invention The present invention is based, at least in part, on the discovery that placing a self-cleaving or bond-forming catalytic RNA, e.g. a ribozyme, in combination with a nucleic acid binding site, e.g. in the stem region of the catalytic RNA, in the 3'UTR of an encoding nucleic acid, e.g. a (selection) marker gene such as eGFP, but before a polyA signal sequence, can be used to control expression of the encoding nucleic acid depending on the presence of a nucleic acid sequence that binds to the nucleic acid sequence binding site of the catalytic RNA.

[0049] RNA-based gene switches are highly desirable for gene therapy, especially for the conditional activation of introduced therapeutic transgenes. Their advantages are, among others, their small size of about 100 nucleotides (nt), their lack of immunogenicity (no proteins involved), and their low complexity (only RNA is required).

[0050] The present invention is based, at least in part, on the discovery that the nucleic acid-responsive ribozyme designs of the present invention are generally applicable. The switching moieties in the nucleic acid-responsive ribozyme designs of the present invention can be easily modified.

[0051] The nucleic acid-responsive ribozyme design of the present invention, i.e., the design of a specific nucleic acid-ribozyme pair, allows the use of any nucleic acid of interest, such as LNA, to control gene expression. LNA is already in clinical trials and is suitable for use in in vivo gene therapy.

[0052] Definitions and general explanations General definition: Methods and techniques useful for carrying out the invention are described, for example, in Ausubel, FM (ed.), Current Protocols in Molecular Biology, Vols. I-III (1997); Glover, ND, and Hames, BD, ed., DNA Cloning: A Practical Approach, Vols. I and II (1985), Oxford University Press; Freshney, RI (ed.), Animal Cell Culture-a practical approach, IRL Press Limited (1986); Watson, JD, et al., Recombinant DNA, Second Edition, CHSL Press (1992); Winnacker, EL, From Genes to Clones; NY, VCH Publishers (1987); Celis, J., ed., Cell Biology, Second Edition, Academic Press (1998); Freshney, RI, Culture of Animal Cells: A Manual of Basic Technique, second edition, Alan R. Liss, Inc., NY (1987).

[0053] The use of recombinant DNA techniques allows the production of derivatives of nucleic acids. Such derivatives can be modified, for example, by substitution, alteration, replacement, deletion or insertion, at individual or several nucleotide positions. Modification or derivatization can be carried out, for example, by site-directed mutagenesis. Such modifications can be easily carried out by those skilled in the art (see, for example, Sambrook, J. et al., Molecular Cloning: A laboratory manual (1999) Cold Spring Harbor Laboratory Press, New York, USA; Hames, BD, and Higgins, SG, Nucleic acid hybridization-a practical approach (1985) IRL Press, Oxford, England).

[0054] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a cell" includes a plurality of such cells and equivalents thereof known to those of skill in the art, and so forth. Similarly, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein. It should also be noted that the terms "comprising," "including," and "having" may be used interchangeably.

[0055] The term "about" refers to a range of ±20% of the numerical value that follows. In certain embodiments, the term "about" refers to a range of ±10% of the numerical value that follows. In certain embodiments, the term "about" refers to a range of ±5% of the numerical value that follows.

[0056] The term "comprising" also includes the term "consisting of."

[0057] The polymerase, for example, moves from the 5' end to the 3' end of the nucleic acid. Thus, the nucleotide sequence of the nucleic acid is written from left to right in the same 5' to 3' direction. This applies in particular to coding nucleic acid sequences, for example in expression cassettes, to reflect the reading direction of the ribosome during the translation process. Thus, the 5' end is the beginning or front or left end of the nucleic acid sequence, and the 3' end is the end or right end of the nucleic acid sequence. The terms 5' end and 3' end are also used to characterize the orientation of coding sequences. Thus, a promoter operably linked to a coding sequence is 5', i.e., in front of, the coding sequence.

[0058] "Operably linked" refers to the juxtaposition of two or more components, where the components so described are in a relationship that allows them to function in their intended manner. For example, a promoter and / or enhancer are operably linked to a coding sequence if they act in cis to control or regulate the transcription of the linked sequence. Generally, but not necessarily, "operably linked" DNA sequences are contiguous, and where necessary to join two protein coding regions, such as a secretory leader and a polypeptide, contiguous and in frame. However, an operably linked promoter is generally located upstream of the coding sequence, but not necessarily adjacent to it. Enhancers need not be contiguous. An enhancer is operably linked to a coding sequence if it increases the transcription of the coding sequence. An operably linked enhancer can be located upstream, within, or downstream of the coding sequence, at a substantial distance from the promoter. A polyadenylation site is operably linked to a coding sequence if it is located at the downstream end of the coding sequence such that transcription proceeds through the coding sequence to the polyadenylation sequence. A translation stop codon is operably linked to an exon nucleic acid sequence if it is located at the downstream end (3' end) of the coding sequence such that translation proceeds through the coding sequence to the stop codon and terminates thereat. Linking is accomplished by recombinant methods known in the art, for example, using PCR methodology and / or by ligation at convenient restriction sites. If no convenient restriction sites exist, synthetic oligonucleotide adaptors or linkers are routinely used.

[0059] Cell-specific definitions: The term "cell clone" as used herein refers to a mammalian cell comprising an exogenous nucleotide sequence capable of expressing a polypeptide, i.e., a recombinant mammalian cell. Such a recombinant mammalian cell is a cell into which one or more exogenous nucleic acid(s) have been introduced, including the progeny of such a cell. In certain embodiments, a cell clone is a mammalian cell comprising a nucleic acid encoding a heterologous polypeptide. Thus, the term "cell clone comprising a nucleic acid encoding a heterologous polypeptide" refers to a recombinant mammalian cell comprising an exogenous nucleotide sequence integrated into the genome of the mammalian cell and capable of expressing a heterologous polypeptide. In certain embodiments, a cell clone is a mammalian cell comprising an exogenous nucleotide sequence integrated into a single site within a locus of the cell's genome. In a preferred embodiment, a cell clone is a mammalian cell comprising an exogenous nucleotide sequence integrated into a single site within a locus of the cell's genome, the exogenous nucleotide sequence comprising a first recombination recognition sequence and a second recombination recognition sequence adjacent to at least one first selection marker, and a third recombination recognition sequence located between the first recombination recognition sequence and the second recombination recognition sequence, and the recombination recognition sequences are all different.

[0060] The term "recombinant cell" as used herein refers to a cell after genetic modification, such as a cell that expresses a heterologous polypeptide of interest and can be used for the production of said heterologous polypeptide on any scale. For example, a "cell clone" refers to a cell in which a coding sequence of a heterologous polypeptide of interest has been introduced into its genome. For example, a "recombinant mammalian cell comprising an exogenous nucleotide sequence" that has been subjected to recombinase-mediated cassette exchange (RMCE), thereby introducing a coding sequence of a polypeptide of interest into the genome of the host cell, is a particular "cell clone".

[0061] As used herein, a "cell clone" refers to a "transformed cell." It includes both the primary transformed cell and progeny derived therefrom, regardless of the number of transfers. The progeny may not be completely identical to the parent cell, for example in nucleic acid content, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included.

[0062] An "isolated cell clone" refers to a cell clone that has been separated from a component of its natural environment.

[0063] An "isolated nucleic acid" refers to a nucleic acid molecule that is separated from a component of its natural environment.

[0064] Targeted embedding: One method for generating recombinant mammalian cell clones used in the methods according to the present invention is recombinant cell clones generated by using targeted integration (TI) for the introduction of the fusion nucleic acid.

[0065] In targeted integration, site-specific recombination is used to introduce exogenous nucleic acid at a specific locus in the genome of a mammalian TI host cell to generate a recombinant cell clone. It is an enzymatic process in which the sequence of the integration site in the genome is exchanged with the exogenous nucleic acid. One system used to perform such a nucleic acid exchange is the Cre-lox system. The enzyme that catalyzes the exchange is Cre recombinase. The sequence to be exchanged is defined by the location of two lox(P) sites in the genome as well as in the exogenous nucleic acid. These lox(P) sites are recognized by Cre recombinase. Nothing more is needed, i.e. no ATP etc. The Cre-lox system was originally discovered in bacteriophage P1.

[0066] The Cre-lox system functions in a variety of cell types, including mammalian, plant, bacterial, and yeast cells.

[0067] In certain embodiments, the fusion nucleic acid is integrated into a mammalian TI host cell by single or double recombinase-mediated cassette exchange (RMCE), resulting in a recombinant mammalian cell clone, such as a recombinant CHO cell clone, into which the fusion nucleic acid is integrated into the genome at a single locus.

[0068] The Cre-LoxP site-specific recombination system is widely used in many biological experimental systems. Cre recombinase is a 38 kDa site-specific DNA recombinase that recognizes 34 bp LoxP sequences. Cre recombinase is derived from bacteriophage P1 and belongs to the tyrosine family of site-specific recombinases. Cre recombinase can mediate both intra- and intermolecular recombination between LoxP sequences. The LoxP sequence consists of an 8 bp non-palindromic core region flanked by two 13 bp inverted repeats. Cre recombinase binds to the 13 bp repeats, thereby mediating recombination within the 8 bp core region. Cre-LoxP-mediated recombination occurs with high efficiency and does not require any other host factors. If two LoxP sequences are positioned in the same orientation on the same nucleotide sequence, Cre recombinase-mediated recombination will excise the DNA sequence located between the two LoxP sequences as a covalently closed circle. If two LoxP sequences are located in opposite orientations on the same nucleotide sequence, Cre recombinase-mediated recombination will reverse the orientation of the DNA sequence located between the two sequences. If two LoxP sequences are on two different DNA molecules and one DNA molecule is circular, Cre recombinase-mediated recombination will result in the integration of the circular DNA sequence.

[0069] A "recombination recognition sequence" (RRS) is a nucleotide sequence that is recognized by a recombinase and is necessary and sufficient for a recombinase-mediated recombination event. The RRS can be used to define the location in a nucleotide sequence where a recombination event is believed to occur.

[0070] The term "matched RRS" indicates that recombination occurs between two RRSs. In certain embodiments, the two matched RRSs are the same.

[0071] In certain embodiments, the RRS can be recognized by Cre recombinase. In certain embodiments, the RRS can be recognized by FLP recombinase. In certain embodiments, the RRS can be recognized by Bxb1 integrase. In certain embodiments, the RRS can be recognized by φC31 integrase.

[0072] In certain embodiments, both RRSs are wild-type LoxP sequences. In certain embodiments, both RRSs are mutant LoxP sequences. In certain embodiments, both RRSs are wild-type FRT sequences. In certain embodiments, both RRSs are mutant FRT sequences. In certain embodiments, the two matching RRSs are different sequences but can be recognized by the same recombinase. In certain embodiments, the first matching RRS is a Bxb1 attP sequence and the second matching RRS is a Bxb1 attB sequence. In certain embodiments, the first matching RRS is a φC31 attB sequence and the second matching RRS is a φC31 attB sequence.

[0073] A "two-plasmid RMCE" strategy or "double RMCE" is used in the method according to the present invention when using a combination of two vectors, i.e., a first vector containing the fusion nucleic acid according to the present invention and a second vector containing an additional selectable marker. For example, but not limited to, the integrated landing site can contain three RRSs, such as an arrangement in which a third RRS ("RRS3") is present between a first RRS ("RRS1") and a second RRS ("RRS2"), where the first vector contains two RRSs that match the first and third RRSs on the integrated exogenous nucleotide sequence, and the second vector contains two RRSs that match the third and second RRSs on the integrated exogenous nucleotide sequence.

[0074] The two-plasmid RMCE strategy involves performing two independent RMCEs simultaneously using three RRS sites. Thus, the landing site of a mammalian TI host cell using the two-plasmid RMCE strategy contains a third RRS site (RRS3) that has no cross activity towards either the first RRS site (RRS1) or the second RRS site (RRS2). The two plasmids to be targeted require the same flanking RRS sites for efficient targeting, with one plasmid (front) flanked by RRS1 and RRS3 and the other (back) flanked by RRS3 and RRS2. In addition, two selectable markers are also required in two-plasmid RMCE. One selectable marker expression cassette was split into two parts. The front plasmid contains a promoter followed by a start codon and an RRS3 sequence. The back plasmid lacks a start codon (ATG) and has an RRS3 sequence fused to the N-terminus of the selectable marker coding region. It may be necessary to insert additional nucleotides between the RRS3 site and the selection marker sequence to ensure in-frame translation of the fusion protein, i.e., operative linkage. Only when both plasmids are correctly inserted will the complete expression cassette of the selection marker be assembled, thus conferring resistance to the respective selection agent to the cells.

[0075] Two-plasmid RMCE involves a recombinase-catalyzed double recombination crossover event between two heterospecific RRSs in a target genomic locus and a donor DNA molecule. Two-plasmid RMCE is designed to introduce copies of the combined DNA sequences from the front and back vectors into a predetermined locus of the mammalian TI host cell genome. RMCE can be performed such that no sequences of the prokaryotic vector are introduced into the mammalian TI host cell genome, thus reducing and / or preventing unwanted triggering of the host's immune or defense mechanisms. The RMCE procedure can be repeated with multiple DNA sequences.

[0076] In certain embodiments, targeted integration is achieved by two RMCEs, where two different DNA sequences, each flanked by two heterospecific RRSs, are both integrated into a predetermined site in the genome of the corresponding RRS of the mammalian TI host cell. In certain embodiments, targeted integration is achieved by multiple RMCEs, where DNA sequences from multiple vectors, each integrated sequence flanked by two heterospecific RRSs, are all integrated into a predetermined site in the genome of the mammalian TI host cell. In certain embodiments, a selection marker may be partially encoded in a first vector and partially encoded in a second vector, such that expression of the selection marker is only possible if both are correctly integrated by double RMCE.

[0077] An exemplary mammalian TI host cell suitable for use in the methods according to the invention is a CHO cell with a landing site integrated at a single site within a locus in its genome, the landing site containing three heterospecific loxP sites for DNA recombination mediated by Cre recombinase.

[0078] In this example, the heterospecific loxP sites are L3, LoxFas, and 2L (see, e.g., Lanza et al., Biotechnol. J. 7 (2012) 898-908; Wong et al., Nucleic Acids Res. 33 (2005) e147), where L3 and 2L are adjacent to the 5' and 3' ends of the landing site, respectively, and LoxFas is located between the L3 and 2L sites.

[0079] This organization of the landing sites as outlined in the previous paragraph allows the simultaneous integration of two vectors, for example the so-called front vector with L3 and LoxFas sites and the back vector with LoxFas and 2L sites inside. The functional elements of the selection marker gene, different from those present in the landing sites, can be distributed between both vectors: the promoter and the start codon can be located on the front vector, whereas the coding region and the polyA signal are located on the back vector. Only the correct recombinase-mediated integration of the nucleic acid from both vectors induces resistance to the respective selection agents.

[0080] Typically, a mammalian TI host cell is a mammalian cell that comprises a landing site for integration into a locus in the genome of the mammalian cell, the landing site comprising a first recombination recognition sequence and a second recombination recognition sequence adjacent to at least a first selectable marker, and a third recombination recognition sequence located between the first recombination recognition sequence and the second recombination recognition sequence, and wherein the recombination recognition sequences are all different.

[0081] An exogenous nucleotide sequence is a nucleotide sequence that is not native to a particular cell, but can be introduced into the cell by a DNA delivery method, such as transfection, electroporation, or transformation. In certain embodiments, the mammalian TI host cell comprises at least one landing site that is integrated into one or more integration sites in the genome of the mammalian cell. In certain embodiments, the landing site is integrated into one or more integration sites within a specific locus of the genome of the mammalian cell.

[0082] In certain embodiments, the integrated landing site comprises at least one selection marker. In certain embodiments, the integrated landing site comprises a first RRS, a second RRS, and a third RRS, and at least one selection marker. In certain embodiments, the selection marker is located between the first RRS and the second RRS. In certain embodiments, the two RRSs are adjacent to the at least one selection marker. That is, the first RRS is located 5' (upstream) of the selection marker, and the second RRS is located 3' (downstream) of the selection marker. In certain embodiments, the first RRS is adjacent to the 5' end of the selection marker, and the second RRS is adjacent to the 3' end of the selection marker. In certain embodiments, the landing site comprises a first RRS, a second RRS, and a third RRS, and at least one selection marker is located between the first RRS and the third RRS.

[0083] In certain embodiments, the selection marker is located between the first RRS and the second RRS, and these two adjacent RRSs are different from each other. In certain preferred embodiments, the first adjacent RRS is a LoxP L3 sequence, and the second adjacent RRS is a LoxP 2L sequence. In certain embodiments, the LoxP L3 sequence is located 5' of the selection marker, and the LoxP 2L sequence is located 3' of the selection marker. In certain embodiments, the first adjacent RRS is a wild-type FRT sequence, and the second adjacent RRS is a mutant FRT sequence. In certain embodiments, the first adjacent RRS is a Bxb1 attP sequence, and the second adjacent RRS is a Bxb1 attB sequence. In certain embodiments, the first adjacent RRS is a φC31 attP sequence, and the second adjacent RRS is a φC31 attB sequence. In certain embodiments, the two RRSs are positioned in the same orientation. In certain embodiments, the two RRSs are both oriented in the forward or reverse direction. In certain embodiments, the two RRSs are positioned in opposite directions.

[0084] Host cell: Any mammalian host cell line adapted to grow in suspension can be used to generate recombinant cell clones that can be treated with the methods according to the present invention.

[0085] Examples of useful mammalian host cell lines are human amniotic cells (e.g., CAP-T cells as described in Woelfel, J. et al., BMC Proc. 5 (2011) p. 133); monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney lines (e.g., HEK293 cells or HEK293T cells as described in Graham, F. et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, JP, Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human hepatocytes (HepG2); mouse mammary tumor (MMT060562); TRI cells, e.g., as described in Mather, JP et al., Annals NYAcad. Sci. 383 (1982) 44-68; MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220), and myeloma cell lines, e.g., Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cells suitable for antibody production, see, e.g., Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, in Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0086] In certain embodiments, the mammalian host cell is, for example, a Chinese Hamster Ovary (CHO) cell (e.g., CHO K1, CHO DG44, etc.), a human embryonic kidney (HEK) cell, a lymphoid cell (e.g., Y0, NS0, Sp2 / 0 cell), or a human amniotic cell (e.g., CAP-T, etc.). In a preferred embodiment, the mammalian (host) cell is a CHO cell. Thus, similarly, the cell clone is a CHO cell.

[0087] For TI, any known or future mammalian host cell suitable for TI that contains a landing site described herein integrated at a single site within a genomic locus can be used in the present invention. Such cells are referred to as mammalian TI host cells. In certain embodiments, the mammalian TI host cell is a hamster cell, a human cell, a rat cell, or a mouse cell that contains a landing site as described herein. In a preferred embodiment, the mammalian TI host cell is a CHO cell. In certain embodiments, the mammalian TI host cell is a Chinese Hamster Ovary (CHO) cell, a CHO K1 cell, a CHO K1SV cell, a CHO DG44 cell, a CHO DUKXB-11 cell, a CHO K1S cell, or a CHO K1M cell that contains a landing site described herein integrated at a single site within a genomic locus.

[0088] In certain embodiments, the mammalian TI host cell comprises an integrated landing site, the landing site comprising one or more recombination recognition sequences (RRS). The RRS can be recognized by a recombinase, for example, Cre recombinase, FLP recombinase, Bxb1 integrase, or φC31 integrase. The RRS can be selected, independently of each other, from the group consisting of LoxP sequence, LoxP L3 sequence, LoxP 2L sequence, LoxFas sequence, Lox511 sequence, Lox2272 sequence, Lox2372 sequence, Lox5171 sequence, Loxm2 sequence, Lox71 sequence, Lox66 sequence, FRT sequence, Bxb1 attP sequence, Bxb1 attB sequence, φC31 attP sequence, and φC31 attB sequence. If multiple RRSs must be present, the selection of each sequence is dependent on the other, insofar as non-identical RRSs are selected.

[0089] Combinatorial Library: RMCE-based targeted integration can be used to create combinatorial expression libraries in CHO cells, where each cell contains exactly one fusion nucleic acid. Thus, an exemplary RMCE-based method for generating a recombinant cell library comprising a library of fusion nucleic acids according to the invention includes: a) providing a targeted integration host cell comprising an exogenous nucleotide sequence integrated at a site within a locus of the host cell's genome, the exogenous nucleotide sequence comprising a first recombination recognition sequence and a second recombination recognition sequence adjacent to at least a first selectable marker, and a third recombination recognition sequence located between the first recombination recognition sequence and the second recombination recognition sequence, wherein all of the recombination recognition sequences are different; b) introducing into the cells provided in a) a library of first vectors, each comprising two recombination recognition sequences that match a first recombination recognition sequence and a third recombination recognition sequence on the integrated exogenous nucleotide sequence, where the two recombination recognition sequences are at least (in part) flanked by a randomized fusion nucleic acid and a second selection marker, and a single second vector, each comprising two recombination recognition sequences that match a second recombination recognition sequence and a third recombination recognition sequence on the integrated exogenous nucleotide sequence, where the two recombination recognition sequences are flanked by a further selection marker nucleic acid sequence; c) introducing one or more recombinases either simultaneously with i) the first vector library of b) or sequentially thereafter, one or more recombinases recognize recombination recognition sequences of the first vector and the second vector (optionally, the one or more recombinases perform two recombinase-mediated cassette exchanges), introducing the recombinase; and d) selecting recombinant host cells expressing the second and / or further selection markers; Thereby preparing a recombinant fusion nucleic acid cell library.

[0090] To generate a library of recombinant mammalian cells, e.g., CHO cells, in which each cell expresses a single member of the fusion nucleic acid library, a library of front plasmids containing different members of the fusion nucleic acid library and a single back plasmid containing an additional selection marker are mixed and transfected into a TI host. Mediated by Cre recombinase, the front plasmid is randomly paired with the back plasmid at the target locus. The pool of stably transfected cells is then selected with selectable markers 2 (positive selection) and 3 (negative selection) and an additional selection marker.

[0091] The expression library of cells thereby obtained is subjected to single cell cloning by methods such as limiting dilution, cell sorting or cell printing. The obtained single cells or their clonal progeny can be screened for the desired activation / inactivation by a second nucleic acid.

[0092] Specific embodiments of the present invention Ribozymes are a class of RNA molecules that can catalyze the cleavage or formation of covalent bonds in nucleic acid molecules. Typically, ribozymes are self-cleaving.

[0093] At least nine classes of naturally occurring small self-cleaving ribozymes have been described so far: hammerhead, hairpin, human hepatitis-δ, Varkud satellite, GlmS, twister, twister sister, hitche and pistol ribozymes (de la Pena et al., Molecules, 22 (2017) 78).

[0094] For example, the hammerhead ribozyme class contains a core catalytic site surrounded by three stem and tertiary loop-loop interactions that are required for the high self-cleavage activity of ribozyme function (Martick and Scott, Cell, 126 (2006) 309-320).

[0095] However, a single nucleotide exchange (A→G) in the catalytic core inactivates the self-cleavage activity of the hammerhead ribozyme. The difference in GFP expression as determined by FACS is shown in Figure 1 and a schematic diagram of each construct is shown in Figure 2.

[0096] In the method according to the invention, it is possible to select mutant ribozymes that are leaky, i.e., minimally expressed, in the absence of an external stimulus by selecting cells that show the highest expression of the marker gene in the presence of a stimulus and the lowest expression of the marker gene in the absence of a stimulus, and vice versa. Thus, the change in the fold inactivation or activation can be used as a selection criterion. This is illustrated in FIG. 3.

[0097] A "riboswitch" is a regulatory ribonucleic acid sequence, i.e., a part of an mRNA molecule that regulates the translation of said mRNA molecule. It is therefore a cis-acting element. The catalytic activity of a riboswitch depends on the binding of an effector molecule. This causes the three-dimensional structure of the riboswitch to change, for example, from an inactive form to an active form. A riboswitch is composed of three domains: an aptamer domain, a switching sequence, and an expression platform. In the absence of an effector molecule, the expression platform incorporates the switching sequence into an anti-terminator stem loop (AT) and transcription proceeds through the coding region of the mRNA. In the presence of an effector molecule, the switching sequence is incorporated into the aptamer domain and the expression platform folds into a terminator stem loop (T), interrupting transcription. That is, the expression platform can switch between two different secondary structures in response to the binding of an effector molecule, one of which is transcriptionally active and one of which is transcriptionally inactive. (https: / / www.nature.com / scitable / topicpage / riboswitches-a-common-rna-regulatory-element-14262702 / )

[0098] Self-cleaving RNA molecules are called "ribozymes". A particular class of ribozymes are the "hammerhead ribozymes" (HHRs). Their cleavage activity is strongly influenced by tertiary loop-loop interactions, i.e., the correct tertiary structure.

[0099] In the present specification, the modular ribozyme scaffold from HHR type III, designated as "Env140" by Auslaender et al. (Nature Meth. 11 (2014) 1154), is used as an example. However, based on the teachings provided herein, any other self-cleaving ribozyme can be used in the method according to the present invention.

[0100] Combination of HHR with an RNA aptamer in response to an effector molecule renders the self-cleavage activity of HHR ligand-dependent. HHRs fold into distinct tertiary structures consisting of a three-way junction where stem-loop I / II form specific tertiary interactions required for efficient self-cleavage. Although the catalytic region is highly conserved, the nucleotide composition of the stem-loop varies within individual HHR species, indicating that there are many ways to form the necessary loop-loop interactions that facilitate folding into an active ribozyme conformation.

[0101] The method according to the invention is first exemplified below using the K4 / K19 tetracycline-responsive ribozyme-switch created by Beilstein et al. (ACS Synth. Biol., 15 (2015) 526-534). This is presented only as an example of the method according to the invention and should not be construed as a limitation thereof. The true scope is set forth in the appended claims.

[0102] Although the output measurements were different (luciferase vs. GFP), a 1.9-fold increase was achieved at a concentration of 50 μM tetracycline (FIGS. 4 and 5).

[0103] It is therefore shown that introduction of a trigger-inactivating ribozyme in the 3'UTR between the coding sequence and the polyA signal sequence can be efficiently used to introduce an off-switching of expression of a marker gene in stably transfected cells.

[0104] Another ribozyme is Env140. This ribozyme is described by Auslaender et al. (Nucl. Acids Res., 44 (2016) e94), which is expressly incorporated herein by reference. Figure 2A in combination with Figure 3A of Auslaender et al. shows the structure of Env140. These are reproduced in Figure 6.

[0105] Env140 consists of three stem-loop structures (I, II, III) that form a three-way junction surrounding a conserved catalytic core sequence. Tertiary loop I contacts and tertiary loop II contacts are indicated by red lines. Red arrows point to the cleavage site; italicized nucleotides highlight the inactivating A to G mutation; blue nucleotides represent the ribosome binding site (RBS).

[0106] The 5' to 3' sequence of Env140 is as follows: - a first portion of the stem nucleic acid sequence: TIFF2024543872000002.tif5128-cleavage site, TIFF2024543872000003.tif5128--a first stem-loop nucleic acid sequence (loop I), the 5' portion and the 3' portion of which can form / form a duplex; -GGG GCU GGA CCG CCC C (sequence number 03) - a first portion of a catalytic core sequence, TIFF2024543872000004.tif5128--a second stem-loop nucleic acid sequence (loop II), the 5' and 3' portions of which can / do form a duplex; -GGC CCG CGG AGG GCC (SEQ ID NO: 05) - a second portion of the catalytic core sequence, TIFF2024543872000005.tif11128 - a second portion of the stem nucleic acid sequence that is complementary to (and therefore capable of / will form a duplex with) the first portion of the stem nucleic acid sequence, TIFF2024543872000006.tif5128.

[0107] This corresponds to the sequence of the first nucleic acid, except that either the first portion of the stem nucleic acid sequence or the second portion of the stem nucleic acid sequence is modified to hybridize to an effector nucleic acid, such as an effector LNA.

[0108] A schematic diagram of an exemplary first nucleic acid according to the invention is shown in FIG.

[0109] The effector nucleic acid hybridizes exactly with its 3'-terminal nucleotide to the 5'-terminal nucleotide of the second portion of the stem nucleic acid, i.e., the second nucleic acid (effector nucleic acid) hybridizes only with the first nucleic acid (Env140) of the respective portion of the stem nucleic acid of the first nucleic acid, but not with other portions of the first nucleic acid (Env140). Thus, additional complementary nucleotides outside the first nucleic acid can be added to increase the specificity / binding strength of the second nucleic acid.

[0110] Although the sequences herein are given in a 5' to 3' orientation according to the general convention for presenting nucleic acid sequences, it is clear to those skilled in the art that the second nucleic acid applied has a reverse orientation to be complementary to and hybridize with the first nucleic acid. Thus, the sequence complementary to the first nucleic acid presented in a 5' to 3' orientation must be located at the 3' end of the second nucleic acid presented in a 5' to 3' orientation, and vice versa; the second nucleic acid of SEQ ID NO: 13 is alternatively presented in a 3' to 5' orientation. An exemplary first nucleic acid according to the invention was created by replacing a first portion of the stem nucleic acid sequence of Env140 with the sequence CTG AGG GTA GT (SEQ ID NO: 09) and replacing the corresponding sequence in the second portion of the stem nucleic acid with ACT ACC CTC AG (SEQ ID NO: 10).

[0111] The effector nucleic acid hybridizes to a second portion of the stem nucleic acid sequence and an additional 7 nucleotides outside the Env140 sequence (shown in bold / highlighted in FIG. 7), ie the backbone / polyA sequence.

[0112] For generating each stable cell line by double RMCE, the respective front and back vectors are designed (see FIG. 8). The RNA module corresponds to the first nucleic acid of the present invention.

[0113] Restriction endonuclease sites at the 5' and 3' ends served as linker and cloning sequences.

[0114] Two different stable cell lines have been generated. - Cell line S69 with LNA regulatory sequence and constitutive activation: TIFF2024543872000007.tif11130 - Cell line S70 with constitutive inactivation: TIFF2024543872000008.tif11130

[0115] In the FACS analysis, clear differences can be seen between the two cell lines (and therefore ribozyme forms) with regard to GFP expression (FIG. 9).

[0116] For stable cell lines carrying the inducible / switchable construct as well as the inactive construct, LNA attachment to the stem has been applied to modify enzymatic cleavage, and it can be seen that the fluorescence shifts upon application of LNA (Figure 10).

[0117] LNA Trigger Sequence: ATTGTGCCTGAGGGTAGT (SEQ ID NO: 13)

[0118] Although some nucleotides were used as locked nucleic acids of SEQ ID NO:13, all nucleotides in the oligos were synthesized with a phosphorothioate backbone.

[0119] 12.5 μM LNA was applied to the culture medium and the median fold change in fluorescence was determined 3 and 7 days after application to cell lines containing an active / regulatable ribozyme (S69) and an inactive ribozyme (S70) (FIG. 11).

[0120] These data fully demonstrate the action of the method and nucleic acid according to the invention.

[0121] Accordingly, the invention encompasses at least the following independent aspects and dependent embodiments.

[0122] 1. A first nucleic acid comprising the following sequence: a first portion of the stem nucleic acid sequence, -cleavage site, a first stem-loop nucleic acid sequence (loop I), the 5' and 3' portions of which can / do form a duplex, - a first portion of a catalytic core sequence, a second stem-loop nucleic acid sequence (loop II), the 5'- and 3'-portions of which can / do form a duplex, a second portion of the catalytic core sequence, and - a second portion of the stem nucleic acid sequence which is complementary to (and therefore capable of / will form a duplex with) the first portion of the stem nucleic acid sequence.

[0123] 2. A first nucleic acid comprising the following sequence: a first portion of the stem nucleic acid sequence, -cleavage site, a first stem-loop nucleic acid sequence, the 5' and 3' portions of which form a duplex, designated loop I, - a first portion of a catalytic core sequence, a second stem-loop nucleic acid sequence, the 5' and 3' portions of which form a duplex, designated loop II, a second portion of the catalytic core sequence, and a second portion of the stem nucleic acid sequence that is complementary to and forms a duplex with the first portion of the stem nucleic acid sequence.

[0124] 3. The first nucleic acid according to any one of aspects 1 to 2, wherein the sequence is in the 5' to 3' direction.

[0125] 4. The first nucleic acid according to any one of aspects 1 to 2, wherein the sequence is in the 3' to 5' direction.

[0126] 5. A second nucleic acid that is complementary to at least a portion of the first or second portion of the stem nucleic acid sequence of the first nucleic acid according to any one of aspects 1-2 or any one of embodiments 3-4.

[0127] 6. The second nucleic acid of embodiment 5, wherein binding of the second nucleic acid to the first nucleic acid results in a conformational change in the first nucleic acid.

[0128] 7. The binding of the second nucleic acid to the first nucleic acid results in a conformational change in the first nucleic acid, said conformational change being at least one of the following: - dissociation of a first portion of the first stem sequence from a second portion of the stem sequence and hybridization of one of said portions with a second nucleic acid; - dissociation of loops I and II, which leads to inactivation of catalytic activity, - Association of loops I and II leads to activation of catalytic activity 7. The second nucleic acid according to any one of embodiments 5 to 6,

[0129] 8. A composition comprising a pair of a first nucleic acid and a second nucleic acid, The first nucleic acid has the following sequence from 5' to 3': a first portion of the stem nucleic acid sequence, -cleavage site, a first stem-loop nucleic acid sequence, the 5' and 3' portions of which form a duplex, - a first portion of a catalytic core sequence, a second stem-loop nucleic acid sequence, the 5' and 3' portions of which form a duplex, a second portion of the catalytic core sequence, and a second portion of the stem nucleic acid sequence that is complementary to and forms a duplex with the first portion of the stem nucleic acid sequence; Including, the second nucleic acid is complementary to at least a portion of the first or second portion of the stem nucleic acid sequence, and the portion of the stem nucleic acid that is not complementary to the first or second portion is not complementary to another portion of the first nucleic acid; comprising a pair of a first nucleic acid and a second nucleic acid, wherein binding of the second nucleic acid to the first nucleic acid results in inactivation of the catalytic activity of the first nucleic acid; composition.

[0130] 9. The binding of the second nucleic acid to the first nucleic acid results in a conformational change in the first nucleic acid, said conformational change being at least one of the following: - dissociation of a first portion of the first stem sequence from a second portion of the stem sequence and hybridization of one of said portions with a second nucleic acid; - dissociation of loops I and II, which leads to inactivation of catalytic activity, - Association of loops I and II leads to activation of catalytic activity 9. The composition of embodiment 8, wherein

[0131] 10. The first nucleic acid according to any one of aspects 1-2 or any one of embodiments 3-4; or the composition according to aspect 8 or embodiment 9, wherein the first nucleic acid is a hammerhead ribozyme and the stem is loop III of the hammerhead ribozyme.

[0132] 11. The second nucleic acid according to aspect 5 or any one of embodiments 6, 7 or 10; or the composition according to aspect 8 or any one of aspects 9 to 10, wherein the second nucleic acid is an LNA (locked nucleic acid).

[0133] 12. A fusion nucleic acid comprising: - a coding nucleic acid encoding a selectable marker or a therapeutic protein or a regulatory compound; a nucleic acid comprising a first nucleic acid according to any one of aspects 1 or 2 or any one of embodiments 3, 4, 10 or 11, and a polyadenylation signal sequence operably linked to the encoding nucleic acid; A fusion nucleic acid comprising:

[0134] 13. The fusion nucleic acid according to aspect 12, wherein the encoding nucleic acid encodes a fluorescent protein or an antibody or a regulatory protein.

[0135] 14. A composition comprising the fusion nucleic acid of aspect 12 or embodiment 13 and a second nucleic acid that is complementary to at least a portion of the first or second portion of a stem nucleic acid sequence of the first nucleic acid, wherein the portion of the stem nucleic acid that is not complementary to the first or second portion is not complementary to another portion of the first nucleic acid.

[0136] 15. The composition according to aspect 14, wherein the second nucleic acid is the second nucleic acid according to aspect 5 or any one of embodiments 6 to 7.

[0137] 16. A mammalian cell comprising a first nucleic acid according to any one of aspect 1 or 2, or any one of embodiments 3, 4, 10 or 11, or a fusion nucleic acid according to aspect 12 or aspect 13.

[0138] 17. A method for selecting a pair of a first nucleic acid and a second nucleic acid according to aspect 8 or any one of embodiments 9, 10 or 11, comprising the steps of: - providing a library of fusion nucleic acids according to aspect 12 or embodiment 13, wherein all or part of the stem nucleic acid in the first nucleic acid is randomized, - incorporating members of the library of fusion nucleic acids into a mammalian cell by targeted integration; - a single deposition of the cells after integration to generate a single cell library; - determining the members of the single-cell library that are capable of altering the catalytic activity of the first nucleic acid by the addition of a corresponding library of second nucleic acids according to aspect 5 or any one of embodiments 6, 7 or 11, - Selecting members of the single cell library in which the difference in the expression level of the encoding nucleic acid in the absence and presence of the second nucleic acid is greater than in other cells of the single cell library.

[0139] 18. The method of embodiment 17, wherein the targeted integration is by recombinase-mediated cassette exchange.

[0140] 19. The method of aspect 17 or embodiment 18, wherein the targeted integration is by double recombinase-mediated cassette exchange.

[0141] 20. The first nucleic acid according to any one of aspects 1 or 2, or any one of embodiments 3, 4, 10 or 11, which is within the 3'-UTR of a nucleic acid sequence encoding a selectable marker or a therapeutic protein or a regulatory compound.

[0142] 21. The first nucleic acid according to embodiment 20, wherein the coding nucleic acid codes for a fluorescent protein or an antibody or a regulatory protein.

[0143] 22. The first nucleic acid according to any one of aspects 1 or 2, or any one of embodiments 3, 4, 10 or 11, wherein the first nucleic acid is after the coding portion of the nucleic acid sequence encoding a selectable marker or a therapeutic protein or a regulatory compound and before the operably linked polyA signal sequence.

[0144] 23. The first nucleic acid of any one of aspects 1 or 2, or any one of embodiments 3, 4, 10, 11 or 22, wherein the coding sequence is 5' to the first nucleic acid and the polyA signal sequence is 3' to the first nucleic acid.

[0145] 24. The first nucleic acid according to embodiment 22, wherein the coding nucleic acid codes for a fluorescent protein or an antibody or a regulatory protein.

[0146] 25. The fusion nucleic acid according to aspect 12 or embodiment 13, or the composition according to aspect 14 and embodiment 15, or the mammalian cell according to aspect 16, or the method according to aspect 17 or any one of embodiments 18 or 19, or the first nucleic acid according to any one of embodiments 20 to 24, wherein the coding sequence is in an expression cassette.

[0147] 26. The second nucleic acid according to aspect 5 or any one of embodiments 6, 7 or 10; or the composition according to any one of aspects 8 or 14, or any one of embodiments 9, 10, 11 or 15; or the method according to aspect 17 or any one of embodiments 18 and 19, wherein the second nucleic acid comprises a first portion that is complementary to at least a part of the first portion or the second portion of the stem nucleic acid sequence, and a second portion that is complementary to a nucleic acid sequence that is 5' to the first portion of the stem nucleic acid sequence if the second nucleic acid is complementary to at least a part of the first portion of the stem nucleic acid sequence, or a second portion that is complementary to a nucleic acid sequence that is 3' to the second portion of the stem nucleic acid sequence if the second nucleic acid is complementary to at least a part of the second portion of the stem nucleic acid sequence.

[0148] 27. The second nucleic acid of aspect 5 or any one of embodiments 6, 7, 10 or 26; or the composition of any one of aspect 8 or 14, or any one of embodiments 9, 10, 11, 15 or 26; or the method of aspect 17 or any one of embodiments 18, 19 or 26, wherein the second nucleic acid is complementary to a second portion of the stem nucleic acid sequence.

[0149] 28. The second nucleic acid according to aspect 5 or any one of embodiments 6, 7, 10, 26 or 27; or the composition according to any one of aspects 8 or 14 or any one of embodiments 9, 10, 11, 15, 26 or 27; or the method according to aspect 17 or any one of embodiments 18, 19, 26 or 27, wherein the second nucleic acid comprises or consists of a first portion that is complementary to at least a part of the stem nucleic acid sequence and a second portion that is not complementary to the first nucleic acid sequence.

[0150] 29. The second nucleic acid of aspect 5 or any one of embodiments 6, 7, 10, 26, 27 or 28; or the composition of any one of aspect 8 or 14 or any one of embodiments 9, 10, 11, 15, 26, 27 or 28; or the method of aspect 17 or any one of embodiments 18, 19, 26, 27 or 28, wherein the second nucleic acid is not complementary to the first nucleic acid outside of the stem nucleic acid sequence.

[0151] 30. The second nucleic acid according to aspect 5 or any one of embodiments 6, 7, 10, 26, 27, 28 or 29; or the composition according to any one of aspects 8 or 14 or any one of embodiments 9, 10, 11, 15, 26, 27, 28 or 29; or the method according to aspect 17 or any one of embodiments 18, 19, 26, 27, 28 or 29, wherein the second nucleic acid has a length of 8 to 21 nucleotides.

[0152] 31. The second nucleic acid according to aspect 5 or any one of embodiments 6, 7, 10, 26, 27, 28 or 29; or the composition according to aspect 8 or 14 or any one of embodiments 9, 10, 11, 15, 26, 27, 28 or 29; or the method according to aspect 17 or any one of embodiments 18, 19, 26, 27, 28 or 29, wherein the second nucleic acid has a length of 9 to 20 nucleotides.

[0153] 32. The second nucleic acid according to aspect 5 or any one of embodiments 6, 7, 10, 26, 27, 28 or 29; or the composition according to aspect 8 or 14 or any one of embodiments 9, 10, 11, 15, 26, 27, 28 or 29; or the method according to aspect 17 or any one of embodiments 18, 19, 26, 27, 28 or 29, wherein the second nucleic acid has a length of 11 to 18 nucleotides.

[0154] 33. The second nucleic acid according to aspect 5 or any one of embodiments 6, 7, 10, 26, 27, 28, 29, 30, 31 or 32; or the composition according to any one of aspect 8 or 14 or any one of embodiments 9, 10, 11, 15, 26, 27, 28, 29, 30, 31 or 32; or the method according to aspect 17 or any one of embodiments 18, 19, 26, 27, 28, 29, 30, 31 or 32, wherein the second nucleic acid has a first portion of 8 to 13 nucleotides that is complementary to the stem nucleic acid sequence of the first nucleic acid and a second portion of 13 to 8 nucleotides that is complementary to the 5' or 3' nucleic acid sequence of the stem nucleic acid sequence of the first nucleic acid.

[0155] 34. The second nucleic acid according to aspect 5 or any one of embodiments 6, 7, 10, 26, 27, 28, 29, 30, 31 or 32; or the composition according to any one of aspect 8 or 14 or any one of embodiments 9, 10, 11, 15, 26, 27, 28, 29, 30, 31 or 32; or the method according to aspect 17 or any one of embodiments 18, 19, 26, 27, 28, 29, 30, 31 or 32, wherein the second nucleic acid has a first portion of about 11 nucleotides that is complementary to the stem nucleic acid sequence of the first nucleic acid and a second portion of about 7 nucleotides that is complementary to the 5' or 3' nucleic acid sequence of the stem nucleic acid sequence of the first nucleic acid.

[0156] 35. The second nucleic acid according to aspect 5 or any one of embodiments 6, 7, 10, 26, 27, 28, 29, 30, 31, 32, 33 or 34; or the composition according to any one of aspects 8 or 14 or any one of embodiments 9, 10, 11, 15, 26, 27, 28, 29, 30, 31, 32, 33 or 34; or the method according to aspect 17 or any one of embodiments 18, 19, 26, 27, 28, 29, 30, 31, 31, 33 or 34, wherein all nucleotides of the second nucleic acid have a phosphorothioate backbone.

[0157] 36. The second nucleic acid according to aspect 5 or any one of embodiments 6, 7, 10, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35; or the composition according to any one of aspects 8 or 14 or any one of embodiments 9, 10, 11, 15, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35; or the method according to aspect 17 or any one of embodiments 18, 19, 26, 27, 28, 29, 30, 31, 31, 33, 34 or 35, wherein the second nucleic acid has the sequence ATT GTG CCT GAG GGT AGT (SEQ ID NO: 13).

[0158] 37. The second nucleic acid according to aspect 5 or any one of embodiments 6, 7, 10, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36; or the composition according to any one of aspects 8 or 14 or any one of embodiments 9, 10, 11, 15, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36; or the method according to aspect 17 or any one of embodiments 18, 19, 26, 27, 28, 29, 30, 31, 31, 33, 34, 35 or 36, wherein the second nucleic acid comprises more nucleotides as nucleotides that are complementary to the first part or the second part of the first nucleic acid but not to other parts of the first nucleic acid.

[0159] 38. The first nucleic acid according to any one of aspect 1 or 2 or any one of embodiments 3, 4, 10, 11, 22, 23 or 24; or the composition according to any one of aspect 8 or 14 or any one of embodiments 9, 10, 11, 15, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37; or the method according to aspect 17 or any one of embodiments 18, 19, 26, 27, 28, 29, 30, 31, 31, 33, 34, 35, 36 or 37, wherein the first nucleic acid comprises: as a first part of the stem nucleic acid sequence, the sequence CCU CCU CGU (SEQ ID NO: 01), as cleavage site, the sequence CC (SEQ ID NO: 02), - the sequence of GGG GCU GGA CCG CCC C (SEQ ID NO: 03) as a first stem-loop nucleic acid sequence whose 5' and 3' portions form a double strand; - as the first part of the catalytic core sequence, the sequence GCU GAC GA (SEQ ID NO: 04), - the second stem-loop nucleic acid sequence, the 5' and 3' portions of which form a duplex, has the sequence GGC CCG CGG AGG GCC (SEQ ID NO: 05); - as the second part of the catalytic core sequence, the sequence GAA (SEQ ID NO: 06), - the sequence ACG AGG AGG (SEQ ID NO: 08) as a second portion of the stem nucleic acid sequence, which is complementary to and forms a duplex with the first portion of the stem nucleic acid sequence.

[0160] 39. The first nucleic acid according to any one of aspect 1 or 2 or any one of embodiments 3, 4, 10, 11, 22, 23, 24 or 38, wherein the first portion of the stem nucleic acid sequence has the sequence CTG AGG GTA GT (SEQ ID NO: 09) and the second portion of the stem nucleic acid sequence has the sequence ACT ACC CTC AG (SEQ ID NO: 10); or the composition according to any one of aspect 8 or 14 or any one of embodiments 9, 10, 11, 15, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 or 38; or the method according to aspect 17 or any one of embodiments 18, 19, 26, 27, 28, 29, 30, 31, 31, 33, 34, 35, 36, 37 or 38.

[0161] 40. Use of a first nucleic acid according to any one of aspect 1 or 2 or any one of embodiments 3, 4, 10, 11, 22, 23, 24, 38 or 39, or a composition according to any one of aspect 8 or 14 or any one of embodiments 9, 10, 11, 15, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39, for selective activation or inactivation of expression of an operably linked encoding nucleic acid.

[0162] 41. Use of a first nucleic acid according to any one of aspect 1 or 2 or any one of embodiments 3, 4, 10, 11, 22, 23, 24, 38 or 39, or a composition according to any one of aspect 8 or 14 or any one of embodiments 9, 10, 11, 15, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39, for screening and selection of ribozyme-nucleic acid sequence pairs, in which the nucleic acid sequence activates or inhibits the catalytic activity of the ribozyme.

[0163] 42. A method for selecting a mutant ribozyme based on leakiness or tightness of a regulatory property of a first nucleic acid according to any one of aspect 1 or 2 or any one of embodiments 3, 4, 10, 11, 22, 23, 24, 38 or 39, or a composition according to any one of aspect 8 or 14 or any one of embodiments 9, 10, 11, 15, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39.

[0164] The following examples, sequences and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit or scope of the invention. [Brief description of the drawings]

[0165] [Figure 1] A single nucleotide exchange (A→G) in the catalytic core inactivates the self-cleaving activity of the hammerhead ribozyme; the difference in GFP expression as determined by FACS between active and inactive forms of the ribozyme controls GFP expression. [Diagram 2] A single nucleotide exchange (A→G) in the catalytic core inactivates the self-cleavage activity of the hammerhead ribozyme; schematic representation of the construct. [Diagram 3]An example of a method according to the present invention for selecting mutant ribozymes that are leaky, i.e., minimally expressed, in the absence of an external stimulus, by selecting cells that show the highest expression of the marker gene in the presence of a stimulus and the lowest expression of the marker gene in the absence of the stimulus, and vice versa; fold inactivation or fold activation change is used as the selection criteria. [Figure 4] Methods according to the invention exemplified using the K4 / K19 tetracycline-responsive ribozyme-switch created by Beilstein et al. (ACS Synth. Biol., 15 (2015) 526-534); although the output measurements are different (luciferase vs. GFP), a 1.9-fold increase was achieved at 50 μM tetracycline concentration. [Diagram 5] Activation of GFP at 50 μM tetracycline concentration. [Figure 6] Env140, as described by Auslaender et al. (Nucl. Acids Res., 44(2016)e94), expressly incorporated herein by reference. Reproduction of Figure 2A in combination with Figure 3A in Auslaender et al. [Figure 7] Schematic representation of an exemplary first nucleic acid according to the invention based on Env140. [Figure 8] Schematic diagram of the front and back vectors designed to generate respective stable cell lines by double RMCE for stable expression of an exemplary first nucleic acid according to the present invention. [Figure 9] FACS analysis of GFP expression in the two cell lines and thus the ribozyme forms. [Figure 10] For stable cell lines carrying inducible / switchable constructs as well as inactive constructs, LNA attachment to the stem has been applied to modify enzymatic cleavage, and application of LNA has been shown to shift the fluorescence. [Figure 11] Change in GFP expression following contact with 12.5 μM LNA; median fold change in fluorescence at 3 and 7 days after application to cell lines containing active / regulatable ribozyme (S69) and inactive ribozyme (S70). EXAMPLES

[0166] Description of the embodiment overview cell culture CHO TI host cells (see WO 2019 / 126634) were cultured in 500 mL shake flasks at 37° C. and 5% CO2 in proprietary DMEM / F12-based medium and passaged every 3-4 days.

[0167] choice Pool selection and maintenance was performed in 24 deep-well plate format using a culture volume of 4 mL and standard culture conditions (37° C., 5% CO 2 , 50 mm shaking amplitude at 225 rpm).

[0168] Example 1 Cloning and pool production The production of the targeted gene integration pool followed the protocol established by Ng, D., et al.: (Biotechnol. Prog. 37 (2021) e3140) with minor adjustments.

[0169] For testing and screening, an RNA switch controlling the mRNA degradation of the GFP reporter was cloned into the front plasmid, while a red-shifted iRFP gene was cloned into the back plasmid and used for fluorescence normalization purposes. The expression genes were under the control of a strong promoter (SV40). The RNA switch constructs were cloned downstream of the GFP gene and in front of the bovine growth hormone polyadenylation (bGH-polyA) signal sequence. For each construct, both the complementary and anti-complementary strands were ordered from Microsynth AG (Belgach, Switzerland) as 5'-phosphorylated ssDNA oligonucleotides and annealed by snap cooling at 0.166 μM concentration. The annealed constructs contained EcoRI-EcoRI or EcoRI-SpeI recombination sites and overhangs for direct ligation into the digested front plasmid using EcoRI / SpeI-HF enzymes (NEB).

[0170] Production of targeted integration pools was performed using the SF Cell Line 96-well Nucleofector (商標) Electroporation was performed using the 96-well Shuttle instrument (Lonza Group Ltd) according to the manufacturer's instructions. Briefly, 2 μg of front plasmid, 2 μg of back plasmid and 0.8 μg of Cre plasmid solution were added to a 20 μL solution containing approximately 2 × 10E6 host-TI cells resuspended in SF Cell Line Nucleofector-Supplement mix at a ratio of 4-5:1, using the DS167 electroporation program according to the manufacturer's protocol. After electroporation, 80 μL of warm culture medium was added to the electroporation chamber and incubated at 37 °C for 30 min, and the pool was finally transferred to a 24-well deep well plate block for selection and maintenance.

[0171] Selection of doubly integrated cells was performed 2 days after electroporation by adding a concentrated solution of puromycin (Life technologies, A11138-03) and FIAU (Sigma, #SML0632) to the cultured cells, and selection was continued for 15-20 days until a pool viability of >85% was reached, indicating the end of selection. Selected pools were maintained in culture medium containing half the amount of selection marker.

[0172] Example 2 LNA activation assay and flow cytometry To measure LNA-induced activation of GFP expression, stably integrated pools expressing active and inactive ribozyme constructs (S69 and S70) were seeded at 50,000 cells / well in 200 μL medium in 96-well microplates with flat-bottom circular wells and incubated for 3-7 days in standard static cell culture conditions with or without the addition of 12.5 μM LNA oligonucleotides, after which 70 μL aliquots were removed and analyzed by flow cytometry. Flow cytometry was performed on a 4-6 color BD FACSCanto™ or FACS Celesta™ System using the FITC channel for assessment of GFP expression and the APC channel for iRFP expression. Populations of cells were gated to avoid cell doublets detected by forward and side scatter.

[0173] Sequence Listing TIFF2024543872000009.tif138141

Claims

1. A composition comprising a pair of a first nucleic acid and a second nucleic acid, The first nucleic acid has the following sequence in the 5' to 3' direction: a first portion of the stem nucleic acid sequence, - cleavage site, a first stem-loop nucleic acid sequence, the 5' and 3' portions of which form a duplex, - a first portion of the catalytic core sequence, a second stem-loop nucleic acid sequence, the 5' and 3' portions of which form a duplex, a second portion of the catalytic core sequence, and a second portion of the stem nucleic acid sequence that is complementary to and forms a duplex with the first portion of the stem nucleic acid sequence; Including, the second nucleic acid comprises a first portion that is complementary to at least a portion of the first portion or the second portion of the stem nucleic acid sequence, and a second portion that is complementary to a nucleic acid sequence that is 5' to the first portion of the stem nucleic acid sequence if the second nucleic acid is complementary to at least a portion of the first portion of the stem nucleic acid sequence, or a second portion that is complementary to a nucleic acid sequence that is 3' to the second portion of the stem nucleic acid sequence if the second nucleic acid is complementary to at least a portion of the second portion of the stem nucleic acid sequence; Binding of the second nucleic acid to the first nucleic acid results in inactivation of the catalytic activity of the first nucleic acid. composition.

2. The binding of the second nucleic acid to the first nucleic acid results in a conformational change of the first nucleic acid, the conformational change being at least one of the following: - dissociation of the first portion of the stem nucleic acid sequence from the second portion of the stem nucleic acid sequence and hybridization of one of the portions with the second nucleic acid; - dissociation of the first stem loop and the second stem loop, resulting in inactivation of the catalytic activity The composition of claim 1 ,

3. A composition comprising a pair of a first nucleic acid and a second nucleic acid, The first nucleic acid has the following sequence in the 5' to 3' direction: a first portion of the stem nucleic acid sequence, - cleavage site, a first stem-loop nucleic acid sequence, the 5' and 3' portions of which form a duplex, - a first portion of the catalytic core sequence, a second stem-loop nucleic acid sequence, the 5' and 3' portions of which form a duplex, a second portion of the catalytic core sequence, and a second portion of the stem nucleic acid sequence that is complementary to and forms a duplex with the first portion of the stem nucleic acid sequence; Including, the second nucleic acid comprises a first portion that is complementary to at least a portion of the first portion or the second portion of the stem nucleic acid sequence, and a second portion that is complementary to a nucleic acid sequence that is 5' to the first portion of the stem nucleic acid sequence if the second nucleic acid is complementary to at least a portion of the first portion of the stem nucleic acid sequence, or a second portion that is complementary to a nucleic acid sequence that is 3' to the second portion of the stem nucleic acid sequence if the second nucleic acid is complementary to at least a portion of the second portion of the stem nucleic acid sequence; binding of the second nucleic acid to the first nucleic acid results in inactivation of the catalytic activity of the first nucleic acid; the first nucleic acid is a hammerhead ribozyme, and the stem is loop III of the hammerhead ribozyme; composition.

4. The method of claim 1, wherein the binding of the second nucleic acid to the first nucleic acid results in a conformational change of the first nucleic acid, the conformational change being at least one of the following: - dissociation of the first portion of the stem nucleic acid sequence from the second portion of the stem nucleic acid sequence and hybridization of one of the portions with the second nucleic acid; - dissociation of the first stem loop and the second stem loop, resulting in inactivation of the catalytic activity The composition of claim 3, wherein

5. The composition according to any one of claims 1 to 4, wherein the second nucleic acid is an LNA (locked nucleic acid).

6. A fusion nucleic acid, - an encoding nucleic acid encoding a selectable marker or a therapeutic protein or a regulatory compound, in one preferred embodiment a fluorescent protein or an antibody or a regulatory protein, a nucleic acid comprising a first nucleic acid according to any one of claims 1 to 4, and a polyadenylation signal sequence operably linked to said encoding nucleic acid A fusion nucleic acid comprising:

7. A mammalian cell comprising the first nucleic acid according to any one of claims 1 to 4.

8. A method for selecting a pair of a first nucleic acid and a second nucleic acid according to any one of claims 1 to 4, comprising the steps of: - providing a library of fusion nucleic acids according to claim 6, wherein all or part of the stem nucleic acids in the first nucleic acid according to the present invention are randomized; - incorporating members of said fusion nucleic acid library into mammalian cells by targeted integration; - depositing the cells once after said integration to generate a single cell library; - determining members of said single-cell library that are capable of altering said catalytic activity of said first nucleic acid by the addition of a corresponding second nucleic acid library; - selecting members of said single cell library in which the difference in expression level of said encoding nucleic acid in the absence and presence of said second nucleic acid is greater than in other cells of said single cell library; A method comprising:

9. 9. The method of claim 8, wherein the targeted integration is by recombinase-mediated cassette exchange.

10. The method of any one of claims 8 to 9, wherein the targeted integration is by double recombinase-mediated cassette exchange.

11. A pair of a first nucleic acid and a second nucleic acid, The first nucleic acid has the following sequence in the 5' to 3' or 3' to 5' direction: a first portion of the stem nucleic acid sequence, - cleavage site, a first stem-loop nucleic acid sequence, the 5' and 3' portions of which form a duplex, - a first portion of the catalytic core sequence, a second stem-loop nucleic acid sequence, the 5' and 3' portions of which form a duplex, a second portion of the catalytic core sequence, and a second portion of the stem nucleic acid sequence that is complementary to and forms a duplex with the first portion of the stem nucleic acid sequence; Including, the second nucleic acid comprises a first portion that is complementary to at least a portion of the first portion or the second portion of the stem nucleic acid sequence, and a second portion that is complementary to a nucleic acid sequence that is 5' to the first portion of the stem nucleic acid sequence if the second nucleic acid is complementary to at least a portion of the first portion of the stem nucleic acid sequence, or a second portion that is complementary to a nucleic acid sequence that is 3' to the second portion of the stem nucleic acid sequence if the second nucleic acid is complementary to at least a portion of the second portion of the stem nucleic acid sequence; The binding of the second nucleic acid to the first nucleic acid results in a conformational change of the first nucleic acid, the conformational change being at least one of the following: - dissociation of the first portion of the stem nucleic acid sequence from the second portion of the stem nucleic acid sequence and hybridization of one of the portions with the second nucleic acid; - dissociation of the first stem loop and the second stem loop, resulting in inactivation of the catalytic activity - association of said first stem loop and said second stem loop resulting in activation of said catalytic activity A pair of a first nucleic acid and a second nucleic acid, 12. A composition comprising a fusion nucleic acid and a second nucleic acid, The fusion nucleic acid is - an encoding nucleic acid encoding a selectable marker or a therapeutic protein or a regulatory compound, a nucleic acid, comprising a first nucleic acid, and - a polyadenylation signal sequence operably linked to the encoding nucleic acid Including, The first nucleic acid has the following sequence: a first portion of the stem nucleic acid sequence, - cleavage site, a first stem-loop nucleic acid sequence, the 5' and 3' portions of which form a duplex, - a first portion of the catalytic core sequence, a second stem-loop nucleic acid sequence, the 5' and 3' portions of which form a duplex, a second portion of the catalytic core sequence, and a second portion of the stem nucleic acid sequence that is complementary to and forms a duplex with the first portion of the stem nucleic acid sequence; Including, the second nucleic acid comprises a first portion that is complementary to at least a portion of the first portion or the second portion of the stem nucleic acid sequence, and a second portion that is complementary to a nucleic acid sequence that is 5' to the first portion of the stem nucleic acid sequence if the second nucleic acid is complementary to at least a portion of the first portion of the stem nucleic acid sequence, or a second portion that is complementary to a nucleic acid sequence that is 3' to the second portion of the stem nucleic acid sequence if the second nucleic acid is complementary to at least a portion of the second portion of the stem nucleic acid sequence. composition.

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