NZ750917B2 - High throughput cell-based screening for aptamers - Google Patents

Abstract

The invention provides eukaryotic cell-based screening methods to identify an aptamer that specifically binds a ligand, or a ligand that specifically binds an aptamer, using a polynucleotide cassette for the regulation of the expression of a reporter gene where the polynucleotide cassette contains a riboswitch in the context of a 5' intron-alternative exon-3' intron. The riboswitch comprises an effector region and an aptamer such that when the aptamer binds a ligand, reporter gene expression occurs.

Description

W0 25085 PCT/IBZOl7/00'l 'l 13 HIGH HPUT CELL-BASED SCREENING FOR APTAMERS FIELD OF THE INVENTION The invention provides screening methods to identify an aptamer that specifically binds a ligand, or a ligand that specifically binds an aptame-r, in a eukaryotic cell using a polynucleotide cassette for the regulation of the expression of a reporter gene where the polynucleotide cassette contains a riboswitch in the context of a 5’ intron—alternative exon—3' intron. The riboswitch comprises an or region and an aptamer such that when the aptamer binds a ligand, reporter gene expression occurs.

BACKGROUND OF THE INVENTION Splicing refers to the s by which intronic sequence is removed from the nascent pre—messenger RNA (pre—mRNA) and the exons are joined together to form the mRNA. Splice sites are ons between exons and introns, and are de?ned by different consensus sequences at the 5’ and 3’ ends of the intron (i.e., the splice donor and splice acceptor sites, respectively). Alternative pre—mRNA splicing, or alternative splicing, is a widespread s occurring in most human genes containing multiple exons. It is carried out by a large multi-component structure called the spliceosome, which is a collection of small nuclear ribonucleoproteins (snRNPs) and a diverse array of auxiliary proteins. By recognizing various cis regulatory ces, the osome de?nes exon/intron boundaries, removes intronic sequences, and splices together the exons into a final translatable message (i.e., the mRNA). in the case of alternative ng, certain exons can be included or excluded to vary the final coding message thereby changing the resulting sed protein.

The present invention utilizes |igand/aptamer-mediated control of alternative splicing to identify aptamer/ligand pairs that bind in the t of a target eukaryotic cell.

Prior to the present invention, aptamers have been generated t a variety of ligands through in vitro screening, however, few have proved to be effective in cells, highlighting a need for systems to screen aptamers that function in the organism of .

SUMMARY OF THE INVENTION In one aspect, the present invention provides a method for selecting an aptamer that binds a ligand in eukaryotic cells comprising the steps of: (a) providing a library of aptainers, (b) introducing members of the library of aptamers into a polynucleotide cassette for the ligand-mediated expression of a reporter gene to create a library of riboswitches, (c) introducing the library of riboswitches into eukaryotic cells, and (d) contacting the otic cells with a ligand, and (e) measuring expression of the reporter gene, wherein the polynucleotide cassette comprises an alternatively-spliced exon, ?anked by a 5' intron and a 3’ intron, and a riboswitch comprising (i) an or region comprising a stem that includes the 5' splice site of the 3’ intron, and (ii) an aptamer, wherein the alternatively- spliced exon comprises a stop codon that is in— frame with the reporter gene when the alternatively-spliced exon is spliced into the reporter gene mRNA.

In one embodiment, the library of aptamers comprises aptamers having one or more ized nucleotides. In one embodiment, the library of rs comprises aptamers having fully ized sequences. In one embodiment, the library of aptamers comprises rs that are n about 15 to about 200 nucleotides in length. In one embodiment, the library of aptamers comprises aptamers that are between about 30 and about '100 tides in length. [none embodiment, the library amers comprises more than 100,000 aptamers. In one embodiment, the library of aptamers comprises more than 1,000,000 aptamers.

In one embodiment, the ligand is a small molecule. In one embodiment, the small molecule ligand is exogenous to the eukaryotic cell. In another embodiment, the. ligand is a molecule produced by the eukaryotic cell including, e.g., a metabolite, nucleic acid, vitamin, co-I‘actor, lipid, monosaccharide, and second messenger.

In one embodiment, the eukaryotic cell is selected from a mammalian cell, an insect cell, a plant cell, and a yeast cell. In one embodiment, the eukaryotic cell is derived from a mouse, a human, a fly (e.g., l-zila gasrer), a ?sh (e.g., Dania 0) or a nematode worm (e.g., Caenor/m/Jditis elegans).

In one embodiment, the reporter gene is selected from the group consisting of a fluorescent protein, lucil‘erase, B-galactosidase and horseradish peroxidase. In one ment, the reporter gene is a cytokine, a signaling molecule, a growth hormone, an antibody, a regulatory RNA, a therapeutic protein, or a peptide. In one embodiment, the expression of the reporter gene is greater than about 10-fold higher when the ligand specifically binds the aptamer than the reporter gene expression levels when the ligand is absent. In further ments, the expression of the reporter gene is greater than about 20, 50, 100, 200, 500, or 1,000-fold higher when the ligand ically binds the aptamer than the reporter gene expression levels when the ligand is absent.

In one embodiment, the 5’ and 3’ introns are derived from intron 2 of the human B—globin gene. In one embodiment, the 5’ intron comprises a stop codon in- frame with the target gene. In one embodiment, the 5’ and '3’ introns are each ndently from about 50 to about 300 nucleotides in length. In one embodiment, the 5’ and 3' introns are each independently from about 125 to about 240 nucleotides in length. In one embodiment, the 5' and/or 3' introns have been modi?ed to include, or alter the sequence of, an intron splice enhancer, an intron splice enhancer, a 5' splice site, a '3’ splice site, or the branch point sequence.

In one embodiment, the effector region stem of the riboswitch is about 7 to about 20 base pairs in length. In one embodiment, the effector region stem is 8 to 11 base pairs in .

In one embodiment, the alternatively-spliced exon is derived from exon 2 of the human dihydrot'olate reductase gene (DHFR), mutant human Wilms tumor 1 exon 5, mouse calcium/calmodulin-dependent protein kinase II delta exon 16, or SIRTl exon 6. In one embodiment, the alternatively-spliced exon is the modified DHFR exon 2. In one embodiment, the alternatively-spliced exon has been modi?ed in one or more of the group consisting of altering the sequence of an exon splice er, altering the sequence of an exon splice enhancer, adding an exon splice enhancer, and adding an exon splice donor. In one embodiment, the alternatively-spliced exon is synthetic (i.e., not derived from a naturally-occurring exon).

In one embodiment, the library of aptamers is d into a r aptamer y before introducing into the polynucleotide cassettes comprising the steps: (a) providing a randomized aptamer library wherein the aptamers in the y comprise multiple 5’ and 3’ constant regions and one or more randomized nucleotides, W0 25085 PCT/[8201 7/001113 (b) ming a two-cycle PCR using the randomized aptamer library as the template and a ?rst primer and second primer that are complementary to the ' and 3’ constant regions, (0) isolating the products of the two-cycle PCR and (d) PCR amplifying a subset of the ed ts of the cle PCR using multiple of primers complementary to a subset of the unique 5’ and 3’ constant regions.

In one embodiment, the library of riboswitches is divided into one or more sub-libraries of riboswitches before being introduced into the eukaryotic cells. In one embodiment, the method for dividing the riboswitch library into sub-libraries comprises the steps of: (a) introducing a library 01' aptamers into a d comprising a gene regulation polynucleotide cassette to make riboswitch y; (b) introducing the itch library into bacteria (e.g., E. 0011'); and (c) collecting bacterial clones (for example by picking bacterial colonies) and extracting plasmid DNA to obtain plasmid sub-libraries ol' riboswitches (referred to herein as primary sub-libraries); In embodiments, secondary sub-libraries 01' riboswitches are generated from a primary plasmid sub-library ol'riboswitches by introducing a primary sub-library into bacteria, collecting bacterial clones and isolating the plasmid DNA. The primary or secondary sub-library are then introduced into eukaryotic cells, the eukaryotic cells contacted with a ligand, and expression of the reporter gene measured to determine r one or more aptamers in the library bind the ligand in the context of the eukaryotic cell.

In one embodiment, the present ion includes an aptamer that binds a target ligand wherein the aptamer is selected by the above methods. In embodiments of the invention, the r comprises the sequence of SEQ ID NO: 14 to 27. In one embodiment, the aptamer sequence comprises the sequence of SEQ ID NO: 24.

In another , the invention provides a method for selecting a ligand that binds an aptamer in a eukaryotic cell comprising the steps of: (a) providing a library of ligands, (b) providing a polynucleotide cassette for the ligand-mediated expression of a reporter gene, (0) introducing the polynucleotide cassette into the eukaryotic cell, (d) ting individual groups of the eukaryotic cell with members of the library of ligands, and (e) measuring the expression of the reporter gene, wherein the polynucleolide cassette comprises an atively-spliced exon, flanked by a 5' intron and a 3' , and a riboswitch comprising (i) an effector region comprising a stem that includes the 5’ splice site of the 3’ intron, and (ii) an aptamer, wherein the alternatively- spliced exon comprises a stop codon that is in— frame with the reporter gene when the alternatively-spliced exon is spliced into the reporter gene mRNA.

In one embodiment, the ligand is a small molecule. In one embodiment, the small molecule ligand is exogenous to the eukaryotic cell. In another embodiment, the ligand is a molecule ed by the eukaryotic cell including, e.g., a metabolite, c acid, n, co-I'actor, lipid, monosaccharide, and second messenger.

In one embodiment, the eukaryotic cell is selected from a mammalian cell, an insect cell, a plant cell, and a yeast cell. In one embodiment the eukaryotic cell is derived from a mouse, a human, a ?y (e.g., Dmrophila melwmgaster), a ?sh (e.g., Dania rerio) or a nematode worm (e.g., Caenor/mbdifis elegans).

In one embodiment, the reporter gene is selected from the group consisting of a ?uorescent protein, luciferase, B-galactosidase and horseradish peroxidase. In one embodiment the reporter gene is a cytokine, a ing molecule, a growth hormone, an antibody, a regulatory RNA, a therapeutic protein, or a e. In one embodiment, the expression of the er gene is greater than about 10-fold higher when the ligand specifically binds the aptamer than the reporter gene expression levels when the ligand is absent. In further embodiments, the sion of the reporter gene is greater than abOut 20, 50, 100, 200, 500, or 1.000-l‘old higher when the ligand specifically binds the aptamer than the reporter gene expression levels when the ligand is absent.

In one embodiment, the 5’ and 3’ introns are derived from intron 2 of the human B-globin gene. In one embodiment, the 5’ intron comprises a stop codon in-frame with the target gene. In one embodiment, the 5’ and 3’ introns are each independently from about 50 to about 300 nucleotides in length. In one embodiment, the 5’ and '3' introns are each independently from about 125 to about 240 nucleotides in . In one embodiment, the 5' and/or 3' introns have been modi?ed to include, or alter the sequence of, an intron splice enhancer, an exon splice enhancer, a 5' splice site, a 3’ splice site, or the branch point sequence.

In one embodiment, the effector region stem of the riboswitch is about 7 to about 20 base pairs in length. In one embodiment, the effector region stem is 8 to 11 base pairs in length.

In one embodiment, the alternatively-spliced exon is derived from exon 2 of the human dihydrofolate reductase gene (DHFR), mutant human Wilms tumor 1 exon 5, mouse calcium/calmodulin-dependent protein kinase II delta exon 16. or SIRT‘l exon 6. In one embodiment, the alternatively-spliced exon is a modified DHFR exon 2. In one ment, the alternatively-spliced exon has been modified in one or more of the grOup ting of ng the sequence of an exon splice silencer, altering the sequence of an exon splice enhancer, adding an exon splice enhancer, and adding an exon splice donor. In one embodiment, the alternatively-spliced exon is synthetic (i.e., not derived from a lly-occun‘ing exon).

In one ment, the present invention includes a ligand selected by the above methods.

In another aspect the invention provides a method for splitting a randomized aptamer library into smaller aptamer sub-libraries comprising the steps: (a) providing a randomized aptamer library wherein the rs in the y comprise multiple 5’ and '3' constant regions and one or more randomized nucleotides, (b) performing a cle PCR using the randomized aptamer library as the te and first primers and second primers that are complementary to the 5’ and 3' constant regions, (0) isolating the products of the two-cycle PCR, and (d) PCR amplifying a subset of the isolated products of the two-cycle PCR using primers complementary to a subset of the unique 5’ and 3' constant regions.

In one embodiment, the randomized aptamer library comprises aptamers having one or more randomized nucleotides. In one embodiment, the randomized aptamer library comprises more than about 100,000 aptamers. In one embodiment, the randomized aptamer library comprises more than about 1,000,000 aptamers.

In one embodiment, the first or second primer in the two-cycle PCR comprises a label selected from the group consisting of biotin, digoxigenin (DIG), bromodeoxyuridine (BrdU), ?uorophore, a chemical group, 6.g. thiol group, or a chemical group e.g. azides used in Click Chemistry.

BRIEF DESCRIPTION OF THE FIGURES Figure la. Schematic of the riboswitch construct. A truncated beta-globin intron sequence was inserted in the coding sequence of the reporter gene, and a mutant, stop- codon containing DHFR exon 2 ) was placed in the inserted intron, thus forming a exon gene expression platform by which the er gene expression is regulated by inclusion/exclusion of the mDHFR exon. A hairpin/stem structure is fonned including the U1 binding site in the intron downstream (3’) of the mDHFR exon with the ered sequence mentary to the U1 binding site. which blocks the U1 binding, thereby leading to the exclusion of stop-codon containing mDH FR exon and target gene expression.

The r sequence is grafted in between the UI binding site and its complementary sequence, ng the l of hairpin formation by aptamer/ligand g.

Figure lb. Dose responses of ucts with regulatory cassettes containing ent aptamer based riboswitches. Guanine riboswitches induced reporter gene expression by responding not only to guanine but also guanosine treatment.

Figure 10 and 1d. Graph demonstrating that the xpt-Gl7 riboswitch induces luciferase activity upon treatment with guanine analogs.

Figure 1e. Fold induction of lucii'erase activity by xpt-Gl7 riboswitch upon treatment with compounds.

Figure 2. Schematic of a template for generating randomized aptamer sequences. The aptamer sequence (blank bar) is ?anked by constant s (black bars), which contain Bsal site to facilitate. the cloning of aptamer into a gene regulation cassette to generate riboswitches.

Figure 3a to 3e. Schematic description of the method for splitting large randomized r library to smaller sub-libraries.

Figure 3a. The schematic diagram of the two-step gy for splitting a large aptamer library. The first step is to add a unique pair of sequence tags to each r oligonucleotide template. Following the first step, templates with unique tag sequences are amplified using primers that are specific to tagged sequences.

Figure 3b. Three approaches to attaching tag sequences to templates: tag ces incorporated h PCR using primers that contain tag sequences at the 5' end of primers (l); tag sequences attached by ligating single stranded template sequence with single stranded tag sequences by T4 RNA ligase ([1); tag sequences linked to templates by ligating double stranded template sequences with double stranded tag sequence by T4 DNA ligase (III).

Figure 3c. Schematic m of two-cycle PCR. For cycle 1, only reverse primers J R which contain tag sequence at 5’ end. After the first cycle, the newly synthesized strand has a sequence tag at its 5’ end. For cycle 2, biotin labeled forward primer JF is added to the PCR reaction, which can only use the newly synthesized strand as template, thus generating the tes with tag sequences at both 5' and 3’ ends and a biotin molecule at 5’ Figure 3d. Generation of tagged aptamer y. After labeling templates with sequence tags and biotin molecule, streptavidin beads are used to separate the labeled/tagged single stranded templates from the rest of the reaction components through denaturing the oligos and beads washing. Then the tagged templates are amplified and expanded using a mixture of primers (F and R primers) that are specific to the tagged ces, thus ting tagged aptamer library that are ready for uent PCR using a single pair of tag sequence-speci?c primers to generate sub-libraries of the original aptamer library.

Figure 3e. Sub-libraries of aptamers are PCR amplified using the splitting strategy. Aptamer library ([06, generated as in Example '2) was tagged by PCR using 2 forward primers (WI -'2) and 8 reverse primers (JR 1 -8), with template copy number at l, 2.3 or 4.6. The isolated tagged templates were expanded by a mixture of tag-specific s F'l-2 and R1 -8, and the PCR products were subject to PCR with either universal primers (left W0 25085 PCT/[8201 7/001113 panel), single pair of tag-specific primers F1 and R1 (middle , or single pair non- relevant primers of F3 and RI (right . Water was used as blank control for templates.

Figure 4. Sensitivity test on cell-based assay for riboswitch y ing.

Construct xpt—G 17 was mixed with construct SR-mut at different molecular . and the mixed construct DNA was transl'ected into HEK—293 cells and treated with guanine. The [old induction ol‘lucil'erase activity was calculated as lucil‘erase activity induced with guanine divided by luciferase activity obtained without guanine treatment.

Figure 5a. Schematic diagram of construction of a plasmid library containing riboswitch. The single stranded r oligos are ?rst PCR amplified using universal primers to convert single stranded aptamer template to double ed. The double stranded oligos are then digested with Bsal and ligated to Bsal-digested vector to generate constructs with riboswitches. The plasmid DNA is then electroporated into electro-competent DH5a cells. More than 5xlO6 colonies are collected to cover more than 99% of the initial aptamer library (106).

Figure 5b. Schematic diagram of dividing plasmid library of riboswitches to sub-libraries. Plasmid library of riboswitches is transformed into chemically competent DH5a cells. Then transformed bacteria are plated into agar plates. Certain numbers of bacteria] colonies are collected from each individual agar plates and plasmid DNA is extracted from individual colony collection separately. The obtained plasmid DNA from each collection 01‘ colonies forms the Sub-library ol' itch. The dividing approach can be ed until desired size of braries is achieved.

Figure 50. Unique sequence composition of secondary sub-libraries of the riboswitch determined by Next Generation Sequencing. Sequences with more than 12 reads from the sequencing run were considered true sequences.

Figure 5d. Comparison of unique sequence ition between two secondary sub-libraries that are generated from the same primary sub-library P’l S_003. A pie chart indicates the number of unique sequences in each brary and the number of the overlapping sequences between the. two libraries of riboswitches.

Figure 5e. Comparison ofunique sequence. composition between two secondary sub-libraries that are generated from different primary sub-libraries, Pl S_003 and P] S_007, respectively. A pie chart indicates the number of unique sequences in each sub- y and the number 01' the overlapping sequences between the two libraries 01' riboswitch.

Figures 6a and 6b. Plasmid DNA from 6 out of 100 primary sub-libraries (60k) (Figure 6a) or 100 secondary sub-libraries (size of 600) e 6b) was arrayed in the- format of 96-Well plate, and transfected into HEK-293 cells. The fold induction of luciferase activity was calculated as lucil'erase activity induced with e divided by lucii‘erase ty obtained without guanine treatment.

Figure 6c. Riboswitch sub-library ing results using nicotinamide adenine dinucleotide (NAD+) as ligand. The sub-libraries of P2 riboswitch library were arrayed in l format. HEK 293 cells were plated in 96—well plate and transfected with riboswitch y DNAs. Four hours after transfection, cells were treated with 100 pM NAD+. Luciferase activity was measured 20 hours after NAD+ treatment. The fold induction was calculated as the ratio of the luciferase activity obtained from NAD+ treated cells divided by lucil'erase activity obtained from cells without NAD+ treatment. Each dot in the dot plot represents the fold induction from a sub-library or 017 construct as indicated.

Figure 6d. Riboswitch screening s using NAD+ as ligand. Each individual riboswitch construct was arrayed in 96-well format. HEK 293 cells were plated in 96-well plate and transfected with riboswitch constructs. 4 hours after transfection, cells were treated with 100 pM NAD+. Luciferase activity was measured 20 hours after NAD+ treatment. The fold induction was calculated as the ratio of the luciferase activity obtained from NAD+ treated cells divided by luciferase activity obtained from cells t NAD+ treatment. Each dot in the dot plot represents the fold induction from each single itch construct or 017 construct as indicated.

Figure 6e and 6f. uct with new aptamer sequence show enhanced response to NAD+ treatment in a dose dependent manner compared to the G17 riboswitch.

HEK 293 cells were transfected with the 617 or construct #46 with new aptamer ce. 4 hours after transl'ection, cells were treated with different doses of NAD+. Lucilerase activity was measured 20 hours after NAD+ treatment. The fold ion was calculated as the ratio of the luciferase activity obtained from NAD+ d cells divided by luciferase activity obtained from cells without NAD+ ent.

DETAILED DESCRIPTION OF THE INVENTION Methods of screening aptamer/Iigand The present invention provides screening methods to identify aptamers that bind to a , and ligands that bind to an aptamer, in the context of a eukaryotic cell, tissue, or organism. In one aspect, the present invention provides a method for selecting an aptamer that binds a ligand in e-ukaryotic cells comprising the steps of: (a) ing a library of aptamers, (b) introducing members of the y of aptamers into polynucleotide cassettes for the ligand-mediated expression of a er gene, (c) introducing the r containing polynucleotide cassettes into eukaryotic cells, and (d) contacting the eukaryotic cells with a ligand, and (e) measuring expression of the reporter gene.

In r aspect, the invention provides a method for selecting a ligand that binds an aptamer in a eukaryotic cell comprising the steps 01': (a) providing a library of ligands, (b) providing a polynucleotide cassette for the li grind-mediated expression of a reporter gene, (0) introducing the polynucleotide cassette into the eukaryotic cell, (d) contacting individual groups of the eukaryotic cell with members of the y of ligands, and (e) measuring the expression of the reporter gene.

In one embodiment, the invention provides methods to identify aptamers that bind to intracellular molecules comprising the steps of: (a) providing a library of aptamers, (b) ucing members ol~ the y of aptamers into cleotide cassettes for the ligand-mediated expression of a reporter gene, (c) introducing the aptamer containing polynucleotide tes into eukaryotic cells, and (d) measuring expression of the reporter gene.

The screening methods of the present ion utilize the gene regulation polynucleotide cassettes disclosed in PCT/U82016/016234, which is incorporated in its entirety herein by reference. These gene regulation cassettes comprise a riboswilch in the 'l 'l W0 25085 PCT/IBZOI 7/00'l113 context of a 5’ —alternative exon—3’ intron. The gene tion cassette refers to a recombinant DNA construct that, when incorporated into the DNA of a target gene (e.g., a reporter gene), provides the ability to te expression of the target gene by aptamer/ligand ed alternative ng of the resulting pre-mRNA. The gene regulation cassette further comprises a riboswitch containing a sensor region (e.g., an aptamer) and an effector region that together are responsible for sensing the presence of a ligand that binds the aptamer and altering splicing to an alternative exon. These aptamer- driven riboswitches provide regulation of mammalian gene expression at a 2- to ZOOO-fold induction, in responding to treatment with the ligand that binds the aptamer. The unprecedented high dynamic regulatory range of this synthetic riboswitch is used in methods of the present invention to provide screening s for new aptamers against desired types of ligands, as well as for optimal ligands against known and novel aptamers in cells, tissues and sms.

Riboswitch The term "riboswitch" as used herein refers to a regulatory segment of a RNA polynucleotide (or the DNA encoding the RNA polynucleotide). A riboswitch in the context of the present invention contains a sensor region (e.g., an aptamer) and an effector region that er are responsible for sensing the presence of a ligand (e.g., a small le) and altering splicing to an alternative exon. In one embodiment, the riboswitch is recombinant, utilizing polynucleotides from two or more s. The term "synthetic" as used herein in the t of a riboswitch refers to a riboswitch that is not naturally occurring. In one embodiment, the sensor and effector regions are joined by a polynucleotide linker. In one embodiment, the polynucleotide linker forms a RNA stem (Le, a region 01' the RNA polynucleotide that is double-stranded).

A library ol'riboswitches as described herein comprise a plurality of aptmer sequences that differ by one or more nucleotides in the contect of the polynucleotide cassettes for the ligand-mediated expression of a reporter gene. Thus, each r in the library, along with a sensor region, is in the t of a 5' intron—alternative exon—3’ intron as described herein.

Effector region In one embodiment, the effector region comprises the 5' splice site ("5’ 55") sequence of the 3' intron tie, the intronic splice site sequence that is immediately 3' of the alternative exon). The effector region comprises the 5’ ss sequence of the 3’ intron and sequence complimentary to the 5' ss sequence of the 3’ . When the aptamer binds its , the effector region forms a stem and thus prevents ng to the splice donor site at the 3' end of the alternative exon. Under certain conditions (for example, when the aptamer is not bound to its ligand), the effector region is in a context that provides access to the splice donor site at the ‘3' end of the alternative exon leading to inclusion of the alternative exon in the target gene mRNA.

The stem portion of the effector region should be of a sufficient length (and GC'. content) to substantially prevent alternative splicing of the alternative exon upon ligand binding the aptamer, while also allowing access to the splice site when the ligand is not present in sufficient quantities. In embodiments of the invention, the stem portion of the effector region comprises stem sequence in addition to the 5’ ss sequence of the 3’ intron and its complementary sequence. In embodiments of the invention, this additional stem sequence comprises sequence from the aptamer stem. The length and sequence of the stem portion can be ed using known techniques in order to identify stems that allow acceptable background expression of the target gene. when no ligand is present and acceptable expression levels of the target gene when the ligand is present. If the stem is, for example, too long it may hide access to the 5’ ss sequence of the 3' intron in the presence or absence of ligand. If the stem is too short, it may not form a stable stem capable of sequestering the 5’ ss sequence of the 3’ intron, in which case the alternative exon will be spliced into the target gene message in the presence or e of ligand. In one embodiment, the total length of the effector region stem is between about 7 base pairs to about 20 base pairs. In some embodiments, the length of the stem is between about 8 base pairs to about 11 base pairs. In some embodiments, the length of the stem is 8 base pairs to 1‘] base pairs. In addition to the length of the stem, the GC base pair content of the stem can be altered to modify the ity of the stem.

Aptamcr/Ligand The term er" as used herein refers to an RNA polynucleotide (or the DNA encoding the RNA polynucleotide) that speci?cally binds to a ligand or to an RNA polynucleotide that is being screened to identify specific binding to a ligand (Le, a ctive aptanter). A library of aptamers is a collection of prospective aptamers comprising multiple prospective rs having a nucleotide sequence that differs from other members of the y by at least one nucleotide.

The term "ligand" refers to a molecule that is specifically bound by an aptame-r, or to a ctive ligand that is being screened for the ability to bind to one or more aptamers. A library of ligands is a tion of ligands and/or prospective ligands.

In one embodiment, the ligand is a low molecular weight (less than about 1,000 s) molecule including, for example, lipids, monosaccharides, second messengers, co-l'actors, metal ions, other natural products and metabolites, nucleic acids, as well as most therapeutic drugs. In one embodiment, the ligand is a polynucleotide with 2 or more nucleotide bases.

In one embodiment, the ligand is selected from the group consisting of 8- azaguanine, adenosine 5’-monophosphate monohydrate, amphoten'cin B, avermectin Bl, azathioprine, chlormadinone acetate, mercaptopurine, zine hydrochloride, N6- methyladenosine, nadide, progesterone, promazine hydrochloride, pyrvinium pamoate, sull'aguanidine, testosterone propionate, thioguanosine, Tyloxapol and Vorinostat.

In certain embodiments, the methods of the t ion are used to identify a ligand that is an intracellular molecule that binds to the aptamer (i.e., an endogenous ligand) in the polynucleotide cassette thereby causing expression of the reporter gene. For e, cells with a reporter gene ning the polynucleotide cassette for the aptamer/ligand mediated expression, can be exposed to a condition, such as heat, growth, transformation, or mutation, g to changes in cell ing moleCules, metabolites, es, lipids, ions (e.g., Ca2+), etc. that can bind to the aptamer and cause sion 01‘ the reporter gene. Thus, the methods of the present invention, can be used to identify aptamers that bind to intracellular ligands in response to changes in cell state, including, e.g., a change in cell ing, cell metabolism, or ons within the cells. In another embodiment, the present invention is used to identify rs that bind intracellular ligands present in dill'erentiated cells. For example, the methods 01' the present invention may be used to identify ligands or aptamers that bind ligands that are present in induced pluripotent stem cells. In one embodiment, the methods of the present ion can be used to screen for response to cell differentiation in vivo, or physiological changes of cells in vim.

Aptamer ligands can also be cell endogenous components that increase significantly under speci?c physiological/pathological conditions, such as oncogenic transformation - these may include. second messenger molecules such as GTP or GDP, calcium; fatty acids, or fatty acids that are incorrectly metabolized Such as 13-HODE in breast cancer (Flaherty, JT et 21]., Plos One, Vol. 8, e63076, 2013, incorporated herein by reference); amino acids or amino acid metabolites; metabolites in the ysis y that usually have higher levels in cancer cells or in normal cells in metabolic diseases; and cancer-associated molecules such as Ras or mutant Ras n, mutant EGFR in lung cancer, indoleamine-Z,3-dioxygenase (H30) in many types of s. Endogenous ligands e progesterone metabolites in breast cancer as disclosed by JP Wiebe (Endocrine-Related Cancer (2006) 13:717—738, incorporated herein by reference). Endogenous ligands also include metabolites with sed levels resulting from mutations in key metabolic enzymes in kidney cancer such as e, glutathione, kynurenine as disclosed by Minton, DR and Nanus, DM (Nature s, Urology, Vol. '12, 2005, incorporated herein by reference).

The speci?city of the binding of an aptamer to a ligand can be defined in terms of the comparative dissociation constants (Kd) of the aptamer for its ligand as ed to the dissociation constant of the aptamer for unrelated molecules. Thus, the ligand is a molecule that binds to the aptamer with r affinity than to unrelated material.

Typically, the Kd for the aptamer with respect to its ligand will be at least about 10-fold less than the Kd for the aptamer with unrelated molecules. In other embodiments, the Kd will be at least about 20-fold less, at least about 50-fold less, at least about 'lOO-fold less, and at least about 200-fold less. An aptamer will lly be between about l5 and about 200 nucleotides in length. More commonly, an aptamer will be between about 30 and ab0ut 100 nucleotides in length.

The aptamers that can be incorporated as part of the riboswitch and screened by s of the present invention can be a naturally occurring aptamer, or modi?cations thereof, or aptamers that are designed de now or synthetic screened h Systemic evolution of ligands by exponential enrichment (SELEX). Examples of aptamers that bind small molecule ligands include, but are not limited to theophylline, dopamine, sulforhodamine B, and cellobiose kanamycin A, lividomycin, tobramycin. neomycin B, viomycin, chloramphenicol, streptomycin, cytokines, cell surface molecules, and metabolites.

For a review of -rs that recognize small molecules, see, e.g., Famulok, Science 9324-9 (1999) and McKeague, M. & DeRosa, M.C. J. Nuc. Aci. 2012. In another embodiment, the aptamer is a complementary polynucleotide.

] In one embodiment, the. r is prescreened to bind a particular small molecule ligand in vilro. Such methods for designing aptamers include, for example, SELEX. Methods for designing aptamers that selectively bind a small molecule using SELEX are disclosed in, e. g., US. Patent Nos. 5,475,096, 5,270,163, and Abdullah Ozer, et al. Nuc. Aci. '2014, which are incorporated here-in by reference. Modifications of the SELEX process are described in U.S. Patent Nos. 5,580,737 and 5,567,588, which are incorporated herein by nce.

Previous selection techniques for identifying aptamers generally involve preparing a large pool of DNA or RNA molecules of the desired length that contain a region that is randomized or mutagenized. For example, an oligonucleotide pool for aptamer selection might contain a region of 20-100 randomized nucleotides llanked by regions of de?ned sequence that are about 15-25 nucleotides long and useful for the binding of PCR primers. The oligonucleotide pool is ampli?ed using standard PCR techniques, or other means that allow amplification of selected nucleic acid sequences. The DNA pool may be transcribed in vitro to produce a pool of RNA transcripts when an RNA aptamer is desired.

The pool of RNA or DNA oligonucleotides is then ted to a selection based on their ability to bind specifically to the desired ligand. Selection techniques e, for example, affinity chromatography, gh any protocol which will allow ion of nucleic acids based on their ability to bind speci?cally to another molecule may be used. Selection techniques for identifying aptamers that bind small molecules and function within a cell may involve cell based screening methods. In the case of affinity chromatography, the ucleotides are contacted with the target ligand that has been immobilized on a ate in a column or on ic beads. The oligonucleotide is preferably selected for ligand binding in the ce of salt concentrations, temperatures, and other ions which mimic normal physiological conditions. Oligonucleotides in the pool that bind to the ligand are ed on the column or head, and nonbinding sequences are washed away. The ucleotides that bind the ligand are then amplified (after e transcription if RNA transcripts were utilized) by PCR (usually after elution). The selection process is repeated on the selected sequences for a total of about three to ten iterative rounds of the selection procedure. The resulting oligonucleotides are then amplified, cloned, and sequenced using standard ures to identify the ces of the oligonucleotides that are capable of g the target ligand. Once an aptamer sequence has been identified, the aptamer may be further optimized by performing additional rounds of selection starting from a pool of oligonucleotides comprising a mutagenized aptamer sequence.

In one embodiment, the aptamer or aptamer library for use in the present invention comprises one or more aptamers identified in an in vim; aptamer screen. In one embodiment, the aptamers identified in the in vitro aptamer screen have one or more nucleotides randomized to create a prospective aptamer library for use in the methods of the present ion.

The ative exon The alternative» exon that is part of the gene regulation polynucleotide cassette of the present ion can be any polynucleotide seq uence capable of being transcribed to a pre-mRNA and alternatively spliced into the mRNA 01' the target gene. The alternative exon that is part of the gene regulation cassette of the present invention ns at least one sequence that inhibits translation such that when the alternative exon is included in the target gene mRNA. sion of the target gene from that mRNA is prevented or reduced. In a preferred embodiment, the alternative exon contains a stop codon (TGA, TAA, TAG) that is in frame with the target gene when the alternative exon is included in the target gene 1nRNA by splicing. In embodiments, the alternative exon comprises, in addition to a stop codon. or as an alternative to a stop codon, other sequence that s or substantially prevents translation when the alternative exon is incorporated by splicing into the target gene mRNA including, e.g., a NA binding site, which leads to degradation of the mRNA. In one embodiment, the alternative exon comprises a miRNA binding sequence that results in degradation of the mRN A. In one embodiment, the alternative exon encodes a polypeptide sequence which reduces the stability of the protein ning this polypeptide ce. In one embodiment, the alternative exon encodes a polypeptide sequence which directs the protein containing this polypeptide sequence- for degradation.

The basal or background level of splicing of the alternative exon can be optimized by altering exon splice e-r (ESE) sequences and exon splice suppressor (ESS) sequences and/or by introducing ESE or ESS sequences into the alternative exon. Such changes to the sequence 01' the alternative exon can be accomplished using methods known in the art, including, but not limited to site directed mutagenesis. Alternatively, oligonucleotides of a d sequence (e.g., comprising all or part of the alternative exon) can be obtained from conunercial sources and cloned into the gene regulation cassette. Identi?cation of E58 and ESE sequences can be accomplished by methods known in the art, ing, for example using ESE?nder 3.0 (Cartegni, L. et al. ESE?nder: a web resource to identify exonic splicing enhancers. c Acid Research, 2003, 31(13): 3568-3571) and/or other available resources.

In one embodiment, the alternative exon is exogenous to the target gene, although it may be derived from a sequence originating from the organism where the target gene will be expressed. In one embodiment the alternative exon is a synthetic ce. In one embodiment, the alternative exon is a naturally-occurring exon. In r embodiment, the alternative exon is derived from all or part of a known exon. In this t, "derived" refers to the alternative exon containing sequence that is substantially homologous to a naturally ing exon, or a portion thereof, but may contain vari0us mutations. for example, to introduce» a stop codon that will be in frame with the target reporter gene sequence, or to introduce or delete an exon splice enhancer, and/or introduce delete an exon splice suppressor. In one embodiment, the ative exon is derived from exon 2 of the human dihydrofolate reductase gene (DHFR), mutant human Wilms tumor 1 exon 5, mouse calcium/cahnodulin-dependent protein kinase II delta exon 16, or SlRTl exon 6. ' and 3' intronic sequences The alternative exon is flanked by 5' and 3’ intronic sequences. The 5’ and 3’ intronic sequences that can be used in the gene regulation cassette can be any sequence that can be spliced out of the target gene creating either the target gene mRNA or the target gene comprising the alternative exon in the mRNA, depending upon the presence or absence of a ligand that binds the aptamer. The 5' and 3' s each has the sequences necessary for splicing to occur, i.e., splice donor, splice acceptor and branch point ces. In one embodiment, the 5’ and 3’ intronic ces of the gene regulation cassette are derived from one or more naturally occurring s or a n thereof. In one embodiment, the 5' and 3’ intronic sequences are derived from a ted human beta-globin intron 2 (IVS2A). In other embodiments the 5’ and 3’ intronic sequences are derived from the SV40 mRNA intron (used in pCMV-LacZ vector from Clontech), intron 6 of human triose phosphate isomerase (TPI) gene (Nott Ajit, et al. RNA. 2003, 9:6070617), or an intron from human factor [X (Sumiko i et a]. J. Bio. Chem. 1995, 270(10), 5276), the target gene’s own endogenous intron, or any c fragment or synthetic introns (Yi Lai, et a1. Hum Gene Ther. 2006: l7(|0):l036) that contain elements that are sufficient for regulated splicing (Thomas A. Cooper, Methods 2005 (37):331).

In one embodiment, the alternative exon and n'boswitch of the present ion are engineered to be in an endogenous intron of a target gene. That is, the intron (or substantially similar intronic sequence) naturally occurs at that position of the target gene.

In this case, the intronic sequence immediately upstream of the alternative exon is refen‘ed to as the 5’ intron or 5’ intronic sequence, and the intronic sequence immediately ream of the alternative exon is referred to as the '3' intron or 3’ intronic sequence. In this case, the endogenous intron is modified to contain a splice acceptor sequence and splice donor sequence ?anking the 5' and 3’ ends of the alternative exon.

The splice donor and splice acceptor sites in the gene regulation cassette ol' the present invention can be modi?ed to be strengthened or weakened. That is, the splice sites can be modified to be closer to the consensus for a splice donor or acceptor by standard cloning methods, site directed mutagenesis, and the like. Splice sites that are more similar to the splice consensus tend to promote splicing and are thus strengthened. Splice sites that are less similar to the splice consensus tend to hinder splicing and are thus weakened. The consensus for the splice donor of the most common class of s (U2) is NC A G I] G T A/G A G T (where M denotes the exon/intron boundary). The consensus for the splice acceptor is C A G H G (where M denotes the exon/intron boundary). The frequency of particular nucleotides at the splice donor and acceptor sites are described in the art (see, e.g., Zhang, M.Q., Hum Mol Genet. 1988. 7(5):919-932). The strength of 5’ and 3' splice sites can be adjusted to modulate splicing of the alternative exon.

Additional modifications to 5' and ‘3' introns in the gene regulation cassette can be made to modulate ng ing modifying, ng, and/or adding intronic splicing er elements and/or intronic splicing suppressor elements, and/or modifying the branch site sequence.

In one embodiment, the 5' intron has been modified to contain a stop codon that will be in frame with the reporter gene. The 5’ and 3’ ic sequences can also be modi?ed to remove c slice sites, which can be identified with publicly ble software (see, e.g.. Kapustin, Y. et a]. N ucl. Acids Res. 2011. 1-8). The lengths of the 5' and 3’ intronic sequences can be adjusted in order to, for example, meet the size requirements for viral expression constructs. In one embodiment, the 5' and 3' intronic sequences are independently from about 50 to about 300 nucleotides in length. In one embodiment, the 5' and 3' intronic sequences are independently from about 125 to about 240 tides in length.

Reporter genes The screening methods of the present invention utilize a gene regulation cassette that is used to regulate the expression of a target gene (e.g., a reporter gene) that can be expressed in a target cell, tissue or organism. The reporter gene can be any gene whose expression can be used to detect the speci?c interaction of a ligand with the aptamer in the- gene regulation cassette. In one embodiment, the reporter gene encodes a fluorescent protein, including, e.g., a green cent protein (GFP), a cyan ?uorescent protein, a yellow ?uorescent protein, an orange ?uorescent protein, a red ?uorescent protein, or a switchable ?uorescent protein. In r embodiment, the reporter gene encodes a erase enzyme including, e.g., ?re?y erase, Renilla lucil'erase, or secretory Gaussia luciferase. In one embodiment, the reporter gene is B-galactosidase. In one embodiment, the reporter is horseradish peroxidase (HRP). In one embodiment, the reporter gene is selected from the group consisting of a nuclear protein, transporter, cell membrane protein, cytoskeleton protein, receptor. growth hormone, cytokine, signaling molecule, regulatory RNA, antibody, and therapeutic proteins or peptides.

Expression Constructs The present invention contemplates the use of a recombinant vector for uction into target cells a polynucle-otide encoding a reporter gene and containing the gene regulation te ol‘ the present invention. In many embodiments, the recombinant DNA construct of this invention includes additional DNA elements ing DNA ts that provide for the replication of the DNA in a host cell and expression of the target gene in that cell at appropriate levels. The ordinarily skilled artisan appreciates that expression l sequences (promoters, enhancers, and the like) are selected based on their ability to promote sion of the reporter gene in the target cell. "Vector" means a recombinant plasmid, yeast arti?cial chromosome (YAC), mini chromosome, DNA mini-circle or virus (including virus derived sequences) that comprises a polynucleotide to be red into a host cell, either in vitro or in vivo. In one embodiment, the inant vector is a viral vector or a combination of multiple viral vectors. Viral vectors for the aptamer-mediated expression of a reporter gene in a target cell are known in the an and e iral (AV) vectors, adeno—associated virus (AAV) vectors, retroviral and lentiviral vectors, and Herpes simplex type 1 (HSVl) vectors.

Methods for dividing aptamer ies into sub-libraries Another aspect of the present invention provides methods to divide large oligonucleotide libraries into smaller sub-libraries and ches to make cellular assay- screenable plasmid libraries of aptamer-based synthetic riboswitches. One aspect of the invention provides a method for splitting an oligonucleotide library into smaller sub-libraries comprising the steps: (a) providing an oligonucleotide library wherein the oligonucleotides in the library comprise multiple 5’ and 3’ constant regions, (b) performing a cle PCR using the ucleotide library as the template and ?rst primers and second primers that are complementary to the 5’ and 3’ constant regions, to) isolating the products of the two-cycle PCR, and (d) PCR amplifying a subset of the isolated products of the two-cycle PCR using primers complementary to a subset of the unique 5’ and 3' constant regions.

In one embodiment, the oligonucleotide library is a randomized aptamer library containing one or more randomized nucleotides. The r sequences are d by a left and right constant region, which n a restriction site for uent cloning.

In one. embodiment, the first or second primer in the two-cycle PCR comprises a label selected from the group consisting of biotin, genin (DIG), bromodeoxyuridine (BrdU), lluorophore, a chemical group, e.g. thiol group, or a chemical group e.g. azides used in Click Chemistry. These molecules can be linked to the oligonucleotides, and their interacting molecules, such as streptavidin or modi?ed forms of avidin for biotin, antibodies t DIG or BrdU or lluorophore. or a second thiol group to form disul?de, alkyne group for azides, can be. immobilized on a solid e to facilitate the isolation of labeled oligonucleotides.

Once an aptamer library is divided into sub-libraries of aptamers, the aptamers in one or more sub-libraries are introduced into the gene regulation polynucleotide cassette to generate a riboswitch y and screened for ligand binding by the methods provided herein.

Methods for ng riboswitch libraries into sub-libraries In one aspect the t invention pro vides a method for dividing a library of riboswitches into sub-libraries. A library of riboswitches as used herein is a plasmid library comprising a gene regulation polynucleotide cassette, e.g., as described herein and in PCT/USZO'l6/0 l 6234, comprising a plurality of aptamers where individual members of the y comprise aptamer sequences that is different from other members of the library. In embodiments, the aptamers in the library of itches comprise one or more randomized tides. In embodiments, the plasmid riboswitch library was generated from an aptamer brary created by the methods described herein.

The method for dividing the riboswitch library into sub-libraries comprises the steps of: (a) introducing a library 01' aptamers into a plasmid comprising a gene regulation polynucleotide cassette described herein to make a riboswitch library; (b) introducing the riboswitch library into ia (e.g., E. coli); (c) collecting bacterial clones (for example by picking bacterial colonies) and ting plasmid DNA to obtain d sub—libraries of riboswitches (referred to herein as primary sub-libraries); (d) optionally, generating secondary sub-libraries of itches from a primary plasmid sub-library of riboswitches by ucing a primary sub-library into bacteria, collecting bacterial clones and isolating the plasmid DNA. s for introducing sequences into plasmids to generate a library are known in the art as are methods for introducing plasmids into bacteria and obtaining bacterial clones. Bacterial clones containing a member of the d riboswitch library may be collected by plating bacteria and picking individual colonies. Pooled plasmids from these clones form the sub-library. The number of bacterial clones collected determines the size (number of unique members) of the sub-library of riboswitches and multiple sub-libraries may be generated. One or more primary sub-libraries can be further divided to create secondary sub-libraries to further reduce the size of the sub-libraries. The sub-libraries are screened using the s described herein by introducing one or more sub-library into eukaryotic cells, exposing the cells to a ligand of interest, and measuring the expression of the reporter gene from the gene regulation polynucleotide cassette. Increase in reporter gene expression in se to ligand indicates that one or more members of the library comprises an aptamer that binds to the ligand in the context of the riboswitch. Thus, the size 01' the sub- library that can be ed may be determined by the sensitivity of the assay for ing reporter gene sion. In embodiments of the invention, a sub-library comprises about 50 to about 600 unique members (although some members may be repeated in other sub- libraries).

It is to be understood and expected that variation in the principles of invention herein disclosed can be made one of ordinary skill in the art and it is intended that such modi?cations are to be included within the scope of the present ion. All references cited herein are hereby orated by reference in their entirety. The following examples r illustrate the invention, but should not be construed to limit the scope of the invention.

EXAMPLE 1 Mammalian ased screening for r/ligands using splicing-based gene regulating riboswitches.

Procedures: Construction of riboswitches: itches were constructed as described in PCT/USZOl6/Ol6234 (in particular Examples '3 to 6), incorporated herein by nce. A truncated human beta-globin intron sequence was synthesized and inserted in the coding sequence of a ?re?y luciferase gene. A mutant human DHFR exon 2 was synthesized and inserted in the middle of this truncated beta-globin intron sequence using Golden gate cloning gy. Aptamers including xpt-G/Al, ydhl-G/AZ, yxj3, add4, gdg6-G/A5 (citations for the aptamers are incorporated herein by nce) were synthesiaed as oligonucleotides ("oligos") with 4 nucleotide overhang at the 5’ end that are complementary to two different BsaI sites individually (IDT), annealed and ligated to BsaI-digested mDI-IPR-Luci-acceptor VGCLOI'. ] ection: 3.5 x104 HEK 293 cells were plated in a 96-well ?at bottom plate the day before transfection. Plasmid DNA (500 ng) was added to a tube or a 96-well U- bottom plate. Separately, TransIT-293 reagent (Mirus; 1.4 pL) was added to 50 uL Opti- mem 1 media (Life Technologies), and allowed to sit for 5 s at room temperature (RT).

Then, 50 pl. of this diluted transfection reagent was added to the DNA, mixed, and incubated at RT for 20 min. Finally, 7 pL 01‘ this solution was added to a well of cells in the 96-well plate.

Fire?y luciferase assay of cultured cells: 24 hours after media change, plates were removed from the incubator, and equilibrated to RT for several minutes on a lab bench, then aspirated. Glo-lysis buffer (Promega, 100 uL, RT) was added, and the plates maintained at RT for at least 5 minutes. Then, the well contents were mixed by 50 pL tn’turation, and 20 pL of each sample was mixed with 20 pL of bright-glo reagent (Pi-omega) that had been diluted to [0% in glo-lysis buffer. 96 wells were spaced on an opaque white 384-well plate.

Following a 5 min tion at RT, luminescence was measured using a Tecan machine with 500 mSec read time. The luciferase activity was expressed as mean relative light unit (RLU)iS.D., and fold induction was calculated as the rase activity obtained with e treatment divided by luciferase activity obtained without guanine treatment.

Results: ng with luciferase as a reporter gene, a gene expression rm was created by inserting a human ?-globin intron in the middle of the coding sequence of ?re?y luciferase and a mutant stop codon-containing human DHFR exon 2 in the intron n.

The er gene expression is thus lled by the inclusion or exclusion of the mDHFR exon containing a stop codon that is in frame with the reporter gene. In this system, a hairpin structure in the mRNA formed by U1 binding site and an inserted complimentary sequence blocks the inclusion of mDHFR exon, therefore enabling target gene expression e la).

To make the formation of hairpin structure regulatable, thus target gene expression controllable by small molecules, we d either synthetic aptamers (theophylline) or natural aptamers (xpt-G/A, yxj, ydhl-A/G, add-A/G aptamers) or hybrid aptamer gdg6-G/A) to this splicing-based gene regulation platform in between the U1 binding site and its complementary sequence, and generated synthetic riboswitches that regulate gene expression in mammalian cells. By using our splicing-based gene regulation cassette and inserting different rs into our synthetic riboswitch uct, we demonstrated different functional responses to ligand in the context of mammalian cells. Those riboswitches with guanine aptamers responds to guanine as well as guanosine as shown in Figure lb. The xpt- guanine riboswitch. xpt—Gl7 (disclosed in PCT/USZO'l6/016234, see, e.g., SEQ ID NO.:15, incorporated herein by reference), yielded high dynamic range of induction of reporter gene expression in response to ligand with its natural ligand treatment.

Although the l aptamer-based riboswitches have high dynamic range in regulating gene expression in mammalian cells, the nature of the ligands for those l aptamers limits their applicability in vivo. Taking advantage of our highly dynamic gene regulation platform with riboswitches, we ?rst chose a list of guanine analogs that have different chemical groups at N2 position to test their activities on xpt-Gl7 riboswitch. As shown in Figure 1c, at 500 pM concentration, several N2 compounds induced luciferase activity in cells with xpt-Gl7 construct, with N2-Phen0xyacetyl guanine being the most potent fold induction) as shown in Figure 1d. To expand the list of compounds for use as potential ligands, the Prestwick library (a collection of 1280 clinically ed drugs) was used at 100 uM to screen for optimal ligands to activate known aptamers in the context of mammalian cells. As shown in Figure '1e from a preliminary screen, the guanine riboswitch apt-017 responded not only to guanine, but also to 8-azaguanine, Nadide, N6- methyladenosine, Testosterone nate, Adenosine 5’-monophosphate inonohydrate, amphotericin B. Thioguanine, Tyloxapol, Progesteron and Chlorinadinone acetate as shown in Figure 1e, as well as a number er nds as listed in Table l. Intriguingly, some of these compounds that showed activities on xpt-Gl7 riboswitch are structurally very different from guanine or guanosine. The Prestwick library was further screened with other 8 purine riboswitches, and a number of nds that can activate the riboswitches in inducing luciferase activity were obtained (Table '1). These s demonstrate the important usage of the riboswitch system in discovering potential optimal ligands for known aptamer in cellular environment. further highlighting the importance of generating aptamers in the t of the cells within which the riboswitch will be ed to function.

Table l. x ten x [-017 x [-017 Riboswitch Fold Induction 7 _0-9 x t-Gl7 4-9 x-t-Gl7 5-3 "51716.2 XII-61710.3 Riboswitch Fold Induction add-A6 6-5 add-A6 4:2 add-A6 5-9 add-A6 3.5 Fold Induction 1 - 8 42.3 _'— 8 16.2 W0 25085 PCT/[3201 7/001113 Riboswitch Fold Induction add-G6 add-G6 5-0 add-G6 5-1 add-G614"3 Sequences for riboswitches used in the Prestwick library screen are provided below with the stem sequences in capital letters, and the aptamer sequences in lowercase letters: xpt—AS (SEQ ID NO: 1): GTAATGTataatcgcgtggatatggcacgcaagtttctaccgggcaccgtaaatgtccgattACATTAC add-G6 (SEQ TD NO: '2): GTAATGTGtataatcctaatgatatggtttgggagtttctaccaagagccttaaactcttgactaCACAT'TAC add-A6 (SEQ ID NO: 3): GTAATGTGtataatcctaatgatatggtttgggagtttctaccaagagccttaaactcttgattaCACATTAC gdg6-A8 (SEQ ID NO: 4): GTAATGTacagggtagcataatgggctactgaccccgccgggaaacctatttcccgatu-XCATTAC S (SEQ ID NO: 5): GTAATGTacagggtagcataatgggctactgaccccgccgggaaacctatttcccgactACATTAC Ydhl-GS (SEQ ID NO: 6): GTAATGTataacctcaataatatggtttgagggtgtctaccaggaaccgtaaaatcctgactACATTAC Ydhl-A6 (SEQ ID N027): GTAATGTGtataacctcaataatatggtttgagggtgtctacoaggaaccgtaaaatcctgattaCACATTAC yxj-A6 (SEQ ID NO: 8): GTAATGTGtatatgatcagtaatatggtctgattgtttctacctagtaaccgtaaaaaactagattaCACATTAC EXAMPLE 2 Design and synthesis of aptamer library.

Procedure To generate an aptamer library, nucleotides at positions in the aptamer that are identified from crystal structure" 7 as ially involved in ligand binding were randomized. In order to facilitate constructing aptamers into riboswitches, lhe aptamer region was ?anked by constant regions with type 115 ction enzyme (e.g. BsaI) cut sites.

This 153 bp er oligonucleotides containing the aptamer sequence with randomized bases were synthesized by lDT: GACT'TCGGTCTCATCCAGAGAATGAAAAAAAAATCTTCAGTAGAAGGTAATGTA TANNNGCGTGGATATGGCACGCNNGNNNNCNCCGGGCACCGTAAATGTCCGACT ACATTACGCACCATTCTAAAGAATAACAGTGAAGAGACCAGACGG (N ents random nucleotides) (SEQ ID NO: 9). To generate more sequence diversity in the aptamer library, bases at more positions can be randomized. A completely random sequence can also be used to generate the aptamer library.

Results ] As described in Example '1, we have successfully built synthetic riboswitches that regulate mammalian gene expression in responding to small molecule ligand ent.

One of the riboswitches 7 that contains xpt-G guanine aptamer in the splicing-based gene expression cassette. Using lucil'erase as a er gene, we achieved a high dynamic range of gene regulation in response to guanine treatment, with induction fold of 2000 at high concentration of guanine. This unprecedented dynamic range of gene regulation activity by the r/ligand mediated alternative splicing constructs provides a system to screen for aptamers against a desired ligand in mammalian cells, or screen for ligands which bind and te known aptamers.

The xpt-G l7 was selected as a platform to build a starting itch library.

The con?guration gonucleotide sequence was designed to replace the original xpt—G guanine aptamer in the following cloning steps. The nucleotides in the xpt-G guanine aptamer at positions that are known to be critical for guanine binding based on crystallography analysis were randomized. Initially, 10 positions were ized, which generated a library of 1,048,576 aptamer sequences. When more than 10 positions are randomized, libraries larger than 106 sequences can be generated. Though Xpt—G guanine aptamer backbone sequence was used here selectively to randomize, a similar approach can be used to generate aptamer libraries with other known rs, or even completely random sequences without known ligands. Though we chose xpt-Gl7 as platform here, it is important to note that riboswitches with different aptamers, or riboswitches based on mechanisms other than splicing can also be used as a starting platform to generate randomized aptamer sequences.

EXAMPLE 3 Splitting large randomized aptamer library into r sub-libraries of aptamer.

Procedures Oligonucleotides (oligos): JF or JR set of primers have ‘3’ portion sequence complementary to nt s in the synthesized aptamer oligos and 5’ portion ce containing random 20 mer oligo sequences. F or R set of primers are complimentary to the random 20 mer oligo sequences in the .IF or .IR primers. All the primers are synthesized at LDT. The .IF primers were d with biotin at 5’ end (IDT). Synthesized oligos were suspended in DNase and RNase-free water to 100 uM as stock solution, and diluted to desired concentration and quantified using Nanodrop machine or OliGreen method oFisher).

Two-cycle PCR amplification: To add biotinylated oligo-tag, two-cycle PCR amplification was perlormed using Pl'x um PCR kit ing manufacturer’s protocol in a reaction volume of 10 pl. The oligo templates were used at desired copy numbers in PCR reaction (1 to 5 copies per oligo ce in the aptamer library). For the first cycle of amplification, only reverse primers J R were included. The amplification was run at 94°C for 2 minutes, then 94°C for 10 seconds, annealing with a touch-down program from 66°C to 52°C descending at 05°C per minute. Then the polymerase reaction was ed at 68°C for 20 second followed by cooling down to 4°C. Then l0 pl of PCR mixture without template but containing ylated forward primers (biotin-IF) were added to the first cycle PCR tube for the second cycle of amplification using the same PCR steps. The PCR products were ready for incubating with streptavidin-beads.

Isolation of biotinylated oligonucle-otides (oligos): 2x Binding and Washing buffer (BW buffer) was made of 'leE buffer (Ambion) with 2M NaCl. Dynabeads M-270 Streptavidin oFisher) (SA-beads) was blocked with 20 1.1M yeast tRNA solution (Ambion) for IO minutes at room temperature, and washed with lx BW buffer twice, and re- suspended in the same volume of 2xBW buffer as the initial volume of heads used. 50 pl of these treated beads were added to the PCR products together with 100 pl of 2xBW buffer and u] of water. The 200 pl of biotinylated oligos and SA-beads mixture was ted at room temperature for 60 minutes, then beads were. denatured at 95°C for 5 minutes, d ately on ice and washed once with leW bull'er, twice with water for 5 minutes following manufacturer’s protocol. Washing solution was d as much as possible, and the washed heads were ready for PCR reaction.

Oligo sequence tag-specific PCRs: Beads with biotinylated PCR products were added to a total 50 ul of PCR mix using Pfx Platinum PCR kit. The primers are a mixture of F and R set primers. The PCR was ted at 94°C for '2 minutes, subject to 28 cycles of 94°C for 15 seconds, 62°C for '30 s, 68°C for 20 seconds, and an additional extension at 68 °C for 2 min. The PCR product was cooled to 12°C and ready for second round of PCR. For the second round of PCR amplification, 1 pl of the PCR product from the first round of PCR was used as template, and a single pair of F and R primers were used to amplify templates tagged with the complementary sequences. The PCR reaction was preheated at 94°C, and ampli?ed with 25 cycles of 94°C for 15 seconds, 60°C for '30 seconds, 68°C. for 20 seconds, and an additional extension at 68°C for '2 minutes.

Results Although in vitro selection using systematic evolution of ligands by exponential ment (SELEX_)8‘ 9 has been extensively applied to screening large aptamer libraries usually with 1013 to 10L4 sequences for generating numerous aptamers against a wide range of ligands including lites, vitamin col'actors, metal ions, proteins and even whole cells", s for cell-based screens of such large randomized aptamer libraries have not been developed. Moreover, few aptamers generated by SELEX have proved effective in a cellular environment, highlighting the ance of screening rs in the cellular environment where they will be required to function. In order for selected aptamers to work within cells, the binding of the ic aptamer to its ligand must have a functional consequence — which cannot be tested via SELEX, which selects aptamers only based on ligand binding under in vitro conditions. One challenge of developing mammalian cell-based screens for aptamers is the low dynamic gene regulatory range of aptamer-based riboswitches in responding to ligand treatment. In addition to this fundamental limitation, the intrinsic low gene transduction efficiency in ian cells imposes r barrier to screening libraries bigger than 105 sequences. However, we developed synthetic riboswitches that can generate up to several thousand-fold induction of gene expression upon ligand treatment.

This high dynamic range of gene regulation provides the basis of a ased system for screening aptamer/ligands. In order to select aptamers in eukaryotic cells from large r libraries that have high sequence diversity, present invention es multiple strategies and approaches to divide/split large aptamer libraries to smaller sub-libraries that can be cloned into riboswitch cassette to te plasmid libraries that are scr‘eenable through mammalian cell-based assays.

] The gy of splitting large aptanrer libraries is to ?rst add a pair of unique sequences at both the 5' and 3' ends of the synthesized, randomized aptamer oligo sequences (as bed in Example '2). In the second step of this strategy, r sequences attached (tagged/labeled) with unique oligo sequences can he. anrplified using single pair of primers complementary to each pair of ce tags, thus generating different sub-libraries of aptamers (Figure 3a). This two-step process of tagging and PCR can be iterated to split the library to the desired sizes.

To attach unique sequence pairs to the template, we have developed multiple ches (Figure 3b). One approach is to use PCR to incorporate unique sequences to templates (PCR approach). Other approaches include ligating single-strand sequence tag to single-strand template using T4 RNA ligase and ligating by T4 DNA ligase double-strand sequence tags to double-strand templates which are generated by PCR amplification of single-strand oligonucleotide templates (ligation approach). We have developed and tested a two-cycle PCR approach (Figure 3c), and currently are in a s of testing the ligation approaches to adding unique sequences tags.

For using PCR approach to attach sequence tags to generate tagged library of r, one set of PCR printers (JF and .l R) was designed. This set of primers contains the tag sequence in the 5’ portion of the s, and in the ‘3’ portion of primers, the sequence that is complementary to the nt region in the synthesized aptamer oligos. In order to avoid the heterogeneity generated by multi-cycle conventional PCR" using high copy numbers of templates, a two-cycle PCR was ped to attach sequence tag at one end of the template at each cycle (Figure 3c). In this two -cycle PCR, the copy number of the randomized oligo templates was kept minimum to decrease the chance for each template to be ed with nrore than one pair of tag sequences. In order to isolate and purify the tagged templates, we labeled JF primers with biotin molecules, so that magnetic avidin beads can be used to separate biotinylated tagged templates from the rest of the reaction components (Figure 3d). Due to the low copy number of templates we started with the PCR tagging, the isolated biotinylated, tagged templates were ampli?ed and expanded by PCR using a mixture 01' a set of s (J and F s) that are specific to the tag sequences attached to the templates, generating the library of aptamers that have unique pair of sequences at the ends (tagged library of randomized aptamers). This PCR product then serves as template for PCR with a single pair of J and R primers to amplify each tagged te, thus generating the sub-libraries of the original aptanrer library.

In a pilot study where 2 biotin-labeled JF primers (WI and JP?) and 8 reverse JR s (JRl to JR8) were used, resulting in total 16 unique pairs of sequence tags. After generating the tagged library by PCR with templates at 1, 2.3 or 4.6 copies enting 63% 90% or 99% of the initial randomized aptamer library, respectively, different primers were used to test the splitting strategy. As shown on the left panel of Figure 3c, the tagged- templates were amplified by primers complementary to the constant region (universal primers) in the aptamer, which amplify every template in the library. When a single pair of primers (F1 and R1) that are specific to the tag sequence-s added (middle panel), but not the pair of primers (F3 and R1) which was not included in the tagging (right panel) were used, the tagged-templates were amplified at much lower amount ed to the product amplified with universal primer, indicating only a portion (1/16) of the library was ampli?ed.

Thus, the original library was split to smaller braries.

EXAMPLE 4 The ivity of cell-based assay for library screening. ures: DNA constructs: Plasmid DNA constructs containing l7 itch was diluted in DNA construct SR-Mut to different ratio of these two DNA constructs The mixed constructs G17 and SR-mut plasmids DNA were then transfected to HEK 293 cells.

Transfection and luciferase assay were performed as described in e 1.

Results: The sensitivity of cell-based assay for library screening determines how complex or how big the size of aptamer-riboswitch plasmid library can be in order for minimum 1 positive hit to stand out from the rest of the library in the screen. The assay can be for lucilerase activity. lluorescence intensity of ?uorescent protein or growth hormone/cytokine release, depending on the reporter gene chosen, and c elements can be delivered either by transient transl'ection or by viral transduction, e.g. AAV, Adeno Virus, lentivirus etc.

Here, we chose ent transfection to deliver plasmid DNA, and used fire?y luciferase as er gene using xpt—G'I 7 construct as positive itch control vector, an assay that has been extensively tested and used during the development of xpt—Gl 7 riboswitch in mammalian cells. Construct SR-mut was used as negative control vector which has the same genetic elements as xpt-Gl7 construct except that there is no guanine aptamer sequence, therefore does not activate gene expression in response to e treatment.

These two constructs were mixed together to mimic a pooled library situation, though the actual riboswitch library is more complex due to the large molecular diversity generated by nucleotide randomization. Cells transfected with 100% 7 construct DNA yielded 2000-fold induction of luciferase activity upon treatment with 500 pM of guanine when compared to untreated cells. When xpt—G'l7 construct DNA was diluted with SR-mut construct DNA, cells transfected with the mixed DNA showed lower fold ion of luciferase activity. As shown in Figure 3, the fold induction decreased when the ratio of guanine responding xpt-Gl7 construct to non-responding negative SR-mut construct decreased, but still can generate a d induction when there is 1 positive construct out of 2000 molecules, indicating the probability of recovering 1 -responding riboswitch from a mixture 01‘ ligand-nonresponding riboswitches.

For assays other than the above described one, the sensitivities of the assay should be tested to provide guidance for ining the size of the sub-library pools to be screened.

EXAMPLE 5 Construction of pooled aptamer-based riboswitch plasmid library and splitting of larger riboswitch library to smaller screenable sub-libraries.

Procedures: Construction of pooled plasmid library of riboswitches: Ultramer Oligos ning aptamer sequences with randomized bases (see Example 2 for sequence design and composition) were PCR amplified using Platinum Pl'x kit (Invitrogen) to generate deuble stranded DNA fragments, and the generated PCR product was run on 4% agarose gel. The DNA with 153 hp size was rified (Qiagen) and digested with Bsal enzyme (NEB).

The BsaI-digested DNA fragment was then ligated to igested acceptor vector (mDHFR-Luci-Acceptor) as described in Example 1 with a 1:5 ratio of vector to insert using a T4 DNA ligase (Roche). ElectroM AX DHSa-E competent cells were transformed following the manufacturer’s instructions (lnvitrogen) with the ligation product and plated onto agar . Bacterial colonies were pooled and collected, and DNA was extracted to obtain plasmid library of riboswitches (P1).

A similar approach was used to generate a smaller plasmid riboswitch library (P2) in which nucleotide bases at 5 positions in the aptamer were randomized generating a total of 1024 different aptamer sequences (where N s a randomized on): GACTFCGGTCTCATCCAGAGAATGAAAAAAAAATCTTCAGTAGAAGGTAATGTA TANNNGCGTGGATATGGCACGCNNGTTTCTACCGGGCACCGTAAATGTCCGACT ACATTACGCACCATTCTAAAGAATAACAGTGAAGAGACCAGACGG (SEQ ID NO: Transformation of chemically competent DHSOt: 227 pg of plasmid DNA was used to orm 50 it] of ent cells to obtain 1 :10 ratio of plasmid DNA and bacterial cells. The transformed cells were plated onto agar plates after being incubated at 37°C without shaking for 30 minutes, and colonies were pooled and collected for DNA tion using 96-format miniprep kit (Qiagen) to obtain pooled plasmid sub-libraries of riboswitches.

Next Generation Sequencing (N68): The plasmid DNA from secondary or tertiary riboswitch sub-libraries was used as templates, and the following primers were used to generate PCR amplicons that contain the randomized aptamer ces: DHFR_F: 5’- GACTTCGGTCTCATCCAGAGAATGAAAAAAAAATCTTCAGTAGAAGGTAATG-3' (SEQ ID NO: 1 I); IVS_R: 5'- CCGTCTGGTCTCTTCACTGTTATTCT‘TTAGAATGGTGCG—3’ (SEQ ID NO: l2). PCR products were subject to NGS using Illumina MiSeq 2x150bp paired-end platform to generate approximately 700K reads for each sample and subsequent bioinformatics analysis for unique sequence identi?cation and relative abundance ation (Serevice provided by Genewiz). Sequences that showed 12, or more than 12, reads from a sequencing run are ered true sequences.

Results To screen aptamers by a cell-based assay, a plasmid library of riboswitches was generated by cloning the r library into mDHFR—Luci—Acceptor vector (Figure 5a).

The ucts generated contain the same configuration of genetic element as in construct xpt-Gl7, with the only difference being in the aptamer sequences. We started with an r library generated as described in Example 2, a randomized aptamer library comprising of 10° unique sequences. To ensure greater than 99.9% representation of the initial r library, a total of 7.5)t106 colonies, which is 7.5 times the number of sequences in the aptamer library, were ted from agar plates. The plasmid DNA extracted from the collected colonies forms the plasmid library (Pl) consisting of IO" unique riboswitches.

To divide plasmid libraries into sub-libraries that are small enough to be screened using the developed cell-based assay, a strategy was utilized, as outlined in Figure 5b, involving pooling smaller numbers of transformed bacterial colonies and extracting DNA to make plasmid sub-libraries of ?boswitches. This s of dividing plasmid libraries can be performed for several r0u nds to obtain the required size of the sub-libraries in which a single positive event (i.e., speci?c aptamer/ligand g g to reporter gene expression) can be detected based on the ivity of the cell-based assay ped for screening the library, generating primary, secondary or tertiary sub-libraries, respectively.

The size of sub-libraries was calculated as library) = in (fold representation) * N (initial library size)/d ing fold). The "dividing fold" represents the total number of sub- libraries to obtain, and can be any number as desired. Here, we chose 100 as ng fold for the ease of calculation. For the ?rst round of dividing, 6)th6 colonies were collected, which is 6 times the number of riboswitches in the initial d library to obtain greater than 99% representation (l06). For the second round of dividing, 1-fold representation of the primary sub-library was chosen. For the plasmid library with '10611'boswitches we built (Pl ), where , m=6, d=’| 00, the size of each individual sub-library is n=6x‘10". A total of 6x10" bacterial colonies were collected into 100 individual tubes and DNA extracted from each individual tube to generate primary plasmid sub-library ol‘riboswitches (PlS_001 through PlS_100). Using the same strategy and starting with sub-library PlS_001, as an example, the primary sub-library was further divided into 100 even smaller secondary sub- libraries named P'I S_00’l_00l through Pl S_00'l_100. Thus, by performing two rounds of dividing, secondary plasmid braries were generated with 600 riboswitches in each. The sub-libraries of riboswitches can be further divided by the 3rd round of dividing processes to generate tertiary plasmid sub-libraries.

The same approach was used to divide plasmid riboswitch library P2 that contains 1024 unique aptamer sequences. By collecting 100 portions 01' a total 5000 colonies, 100 primary braries PZS_001 to PZS_100 were generated, with each sub- library containing approximately 50 riboswitches.

To determine the composition and the quality of the above generated riboswitch ies, next generation sequencing (NGS) was med on the secondary plasmid sub-libraries of riboswitches that presumably contains 600 riboswitch sequences in each sub-library. F0ur secondary sub—libraries were selected at random where two 01‘ the secondary sub-libraries were generated from the primary sub-library Pl S_003, and the other two ary sub-libraries were generated from primary sub-libraries PlS_007 and P1 S_048, respectively. As shown in Figure 5c, each of the secondary su b-libraries contains approximately 500 or 600 unique sequences, consistent with the number of colonies that were collected for generating ary sub-libraries. A further analysis of the NGS data indicates that between the two ary Sub-libraries (PlS_003_004 and P1 S_003_041) that were ted from the same primary sub-library (Pl S_003_), '39 sequences are contained in both libraries (Figure 5d). When comparing two secondary sub-libraries, Pl S_003_004 and P] S_007_0’2 l that were derived from different y sub-libraries, Pl S_003 and PlS_007, only 3 sequences are shared by both sub-libraries (Figure 5e). These results indicate that using the above described gy, plasmid riboswitch sub-libraries were generated with the desired number of unique sequences that are ready for mammalian cell- based screening.

EXAMPLE 6 Mammalian ased screening for new aptamers t ligands of choice.

As described in Example 5, 100 primary plasmid sub—libraries (P1 S_001 through PlS_100), comprising 60k riboswitches in each pool, were constructed, and 100 secondary plasmid sub-libraries (PIS_001_001 to PIS_001_100) consisting of 600 riboswitches in each were generated by further dividing the primary sub-library PlS_001 using the same strategy. The pooled libraries can be arrayed in 96-well format to facilitate hi gh-through screening. A preliminary screen was performed, using the luciferase reporter assay as described in Example 1, on primary sub-libraries 1 to 006 as well as the sub— libraries of PlS_00 | which is against the initial aptamer sequence, as the , against guanine, tested ligand. The basal level of luciferase activity ted by constructs from either primary sub-libraries or ary sub-libraries varied significantly from that ol’ xpt—G‘l7 construct (data not shown), suggesting that changes in the aptamer sequence by randomizing bases at the selected positions impacted the inclusion/exclusion of the stop codon-containing exon to various extent, therefore affecting the basal rase expression. Following e treatment, alth0ugh cells translected with the 60k primary sub-library PlS_005 generated ‘I .8-fold induction of luciferase ty in comparison to untreated cells (Figure 6a), more than 2-fold induction of rase was not discovered when using guanine as the ligand.

However, 7 of the 100 secondary sub-libraries yielded more than 2- fold induction of lucilerase activity upon guanine treatment, with sub-library PlS_001_075 generating 7.8- fold induction (Figure 6b). In the sensitivity assay described in Example 4, d induction was detected when there was 1 xpt-Gl7 itch among 500 non-ligand responding molecules. Based on this sensitivity test, the result (7.8 fold) from this preliminary screening of the sub-library P1 S_001_075 suggests that there is either 1 itch Out 01' 600 that is onally equivalent to apt-017, or there are several weaker rihoswitches of which the sum of induced luciferase activity is comparable to that of xpt- C317.

To further demonstrate the applicability of the mammalian cell-based screening of the present ion for functional aptamers-containing riboswitches and to er new aptamers with improved activity in responding to a desired ligand, the sub- libraries of plasmid riboswitch library P2 were screened in a 96-well format with NAD+.

The nucleotide bases at the randomized positions in the xpt-guanine aptamer have been linked to riboswitch activity tuning and named tune box (Stoddard, et a]. J Mol Biol. 2013 May 27;425(l0): 1 1). There-fore, change-s of nucleotides at these positions potentially te sequences that have altered riboswitch activity in response to the ligand treatment.

Due to the nature of guanine and its low ability in vivo, NAD+ was chosen as ligand for potential new aptamers. This choice of ligand was based upon the above results from screening the Prestwick compound library against the parental xpt-Gl7 itch, and discovering that NAD+ can regulate the guanine riboswitch, ting approximately 40- fold induction at 100 uM concentration. In an attempt to generate aptamer sequences that have improved riboswitch activity against NAD+, we generated and screened the sub- 'es of P2 (having changes of nucleotides at the above-mentioned 5 positions in the aptamer) using luciferase as reporter gene. As shown in Figure 6c, multiple braries, approximately 50 riboswitches in each, yielded more than 10 fold ion of luciferase expression in response to the treatment of 100 pM NAD+, with one of the Sub-libraries, PZS_002, generating 37 fold induction. whereas a single xpt—Gl7 riboswitch construct showed 32 fold induction in response to the treatment of NAD+ at same concentration.

These screening results indicate that among the approximately 50 riboswitches in the sub-libraries that yielded more than 'I 0 fold induction of luciferase expression, there are riboswitches that can produce lly 'I 0 fold induction, assuming all the riboswitches in the library respond to NAD+ ent. In the sub-library PZS_002 that yielded 37 fold induction, which is higher than the fold induction generated by 017, there is at least 1 riboswitch that functions much better than G17. To further prove this, 96 single constructs derived from brary PZS_002 were screened. As shown in Figure 6d, though multiple ucts lost or produced less induction than 617, a number of single constructs produced higher fold induction than the G17 construct, indicating that nucleotide changes in the tune box ically affect the riboswitch activity in ding to ligand treatment in cells. Using this approach, we identi?ed a number of different tune box sequences (as shown in Table 2), with which the riboswitche-s produced higher fold induction of luciferase than the GI? construct upon NAD+ treatment, with multiple aptamer sequences producing more than 100 fold induction.

Table 2. itches with ed reporter gene expression in mammalian cells in response to ligand, NAD+. Tune box sequences are underlined.

ID N0 14-- ATAATCGCGTGGATATGGCACGCMGTTTCTACCGGGCACCGTAAATGTCCGACTATAACCGCGTGGATATGGCACGCEGTTTCTACCGGGCAC CGTAAATGTCCGACT GCGTGGATATGGCACGCQGTTTCTACCGGGCAC CGTAAATGTCCGACT ATAAGGGCGTGGATATGGCACGCTCGTTTCTACCGGGCACC GTAAATGTCCGACT -_GTAAA—TGTCCGACT --CGTAAATGTCCGACT ATAGTGGCGTGGATATGGCACGCCAGTTTCTACCGGGCACC GTAAATGTCCGACT GCGTGGATATGGCACGCCGGT'TTCTACCGGGCAC CGTAAATGTCCGACT _GTAAA—TGTCCGACT _GTAAATGTCCGACT ATAATGGCGTGGATATGGCACGC?GTTTCTACCGGGCACC GTAAATGTCCGACT 24 #46 ATAATTGCGTGGATATGGCACGC?GTTTCTACCGGGCACC GTCCGACT ATAATTGCGTGGATATGGCACGCG_AGTTTCTACCGGGCACC GTAAATGTCCGACT ‘26 #61 ATAATCGCGTGGATATGGCACGCG_AGTTTCTACCGGGCACC GTAAATGTCCGACT 27 #69 GCGTGGATATGGCACGC?GTTTCTACCGGGCACC GTAAATGTCCGACT One of the new constructs, #46, was l‘unher tested. As shown in Figure 6e and Figure 61‘, new construct #46 responded to NAD+ treatment in a dose-dependent manner and showed Superior impr0vement in the level of induced reporter gene expression as well as in the induction [old when compared with G17 construct. The new constructs also have improved gene regulation in response to guanine treatment (data not shown).

Thus, the present invention provides an approach where a relatively large riboswitch library can be divided into smaller riboswitch sub-library that is screenable through a mammalian ased assay. Moreover, from the riboswitch library, new ces that have improved riboswitch activities in mammalian cells were discovered.

References: l. Mandal, Maumjta, Benjamin Boese, Jeffrey E. Barrick, Wade C. Winkler, and Ronald R. Breaker. "Riboswitches Control Fundamental Biochemical Pathways in Bacillus Subtilis and Other Bacteria." Cell 'l 13, no. 5 (May 30, 2003): 577—86. [015 I] 2. , Maumita, and Ronald R. Breaker. "Adenine Riboswitches and Gene Activation by Disruption of a Transcription Terminator." Nature Structural & Molecular Biology ’1 I, no. ’1 (January 2004): 29—35. doi:10.l 038/nsmb7‘10. 3. Mulhbacher, Jerome, and Daniel A. Lalontaine. "Ligand ition Determinants of Guanine Riboswitches." Nucleic Acids Research 35, no. 16 : 5568— 80. doi:10.1093/nar/gkn1572. 4. Serganov, Alexander, Yu-Ren Yuan, Olga Pikovskaya, Anna Polonskaia, Lucy Malinina, Anh Tuan Phan, Claudia Hobartner, Ronald Micura, Ronald R. Breaker, and Dinshaw J. Patel. "Structural Basis for Discriminative Regulation of Gene Expression by Adenine- and Guanine—Sensing mRNAs." Chemistry & Biology 11, no. 12 ber 2004): 1. doi:10.1016/j.chembiol.2004.11.018.

. Edwards, Andrea L., and Robeit T. Batey. "A Structural Basis for the Recognition of 2’-deoxyguanosine by the Purine Riboswitch." Journal of Molecular Biology 385, no. 3 (January 23, 2009): 938—48. doi:l0.'10'l6/j.jmb.2008.'l0.074. 6. Batey, Robert T.. Sunny D. Gilbeit, and Rebecca K. Montange. ture of a Natural e-Responsive Riboswitch Complexed with the Metabolite Hypoxanthine." Nature 432, no. 7015 (November 18, : 411-15. doi:10.1038/nature03037. 7. Serganov, Alexander, Yu-Ren Yuan, Olga Pikovskaya, Anna Polonskaia, Lucy Malinina, Anh Tuan Phan, Claudia Hobartner, Ronald Micura, Ronald R. Breaker, and w J. Patel. "Structural Basis for Discriminative Regulation of Gene Expression by Adenine- and Guanine-Sensing mRNAs." Chemistry & Biology l I, no. l2 (December 2004): ] . doi:]O.l0l6/j.chembiol.2004.11.018. 8. Ellington, A. D., and J. W. Szostak. "In Vitro Selection of RNA Molecules That Bind Speci?c Ligands." Nature 346, no. 6287 (August 30, 1990): 818—22. doi:10.1038/3468l8a0. 9. Tuerk, C., and L. Gold. "Systematic Evolution of Ligands by Exponential Enrichment: RNA Ligands to iophage T4 DNA rase." Science (New York, NY.) 249, no. 4968 (August 3, 1990): 505—10.

. Ozer, Abdullah, John M. Pagano, and John T. Lis. "New Technologies e Quantum Changes in the Scale, Speed, and Success of SELEX Methods and Aptamer Characterization." Molecular Therapy. c Acids '3 (2014): 61 83. doi:] 0.1038/mtna.2014.34. 'l ‘l. Kebschull, Justus M., and Anthony M. Zador. "Sources of PCR-lnduced Distortions in High-Throughput Sequencing Data Sets." Nucleic Acids Research 43, no. 21 (December 2, 2015): e143. doi:10.1093/nar/gkv7l7.

Claims (15)

Claims

1. An in vitro method for selecting an aptamer that binds a ligand in eukaryotic cells comprising the steps of: (a) providing a library of aptamers, (b) introducing the members of the library of aptamers into a cleotide cassette for the ligand-mediated expression of a reporter gene to create a library of riboswitches, (c) introducing the library of riboswitches into eukaryotic cells, and (d) contacting the eukaryotic cells with a ligand, and (e) measuring expression of the reporter gene, wherein the polynucleotide cassette comprises an alternatively-spliced exon, flanked by a 5' intron and a 3' intron, and a riboswitch comprising (i) an effector region comprising a stem that es the 5' splice site sequence of the 3' intron, and (ii) an aptamer, wherein the alternatively-spliced exon comprises a stop codon that is in-frame with the reporter gene when the alternatively-spliced exon is spliced into the reporter gene mRNA.

2. The method of claim 1, wherein the y of aptamers is divided into a smaller aptamer library before introducing into the polynucleotide tes sing the steps: (a) providing a randomized aptamer y wherein the rs in the library comprise le 5' and 3' constant regions and one or more randomized nucleotides, (b) performing a two-cycle PCR using the randomized aptamer library as the template and a first primer and second primer that are complementary to the 5' and 3' constant regions, the primers each including one of a plurality of tag sequences, (c) ing the products of the two-cycle PCR, and (d) PCR amplifying a subset of the isolated products of the two-cycle PCR using primers complementary to a subset of the 5' and 3' tag sequences.

3. The method according to claim 1 or claim 2, wherein the library of riboswitches is divided into one or more sub-libraries of riboswitches before being introduced into the eukaryotic cells.

4. The method according to claim 3, wherein the library of riboswitches is subdivided into sub-libraries comprising the steps of: (a) introducing the riboswitch library into bacteria; (b) collecting bacterial clones and extracting plasmid DNA to obtain plasmid sub-libraries of riboswitches to generate one or more primary sublibraries.

5. The method ing to claim 4, further comprising the step of generating secondary sub-libraries of riboswitches from a primary plasmid sub-library of riboswitches by ucing a primary sub-library into ia, collecting bacterial clones and isolating the plasmid DNA.

6. An in vitro method for ing a ligand that binds an r in a eukaryotic cell comprising the steps of: (a) providing a library of ligands, (b) providing a polynucleotide cassette for the ligand-mediated sion of a reporter gene, (c) introducing the polynucleotide cassette into the eukaryotic cell, (d) ting individual groups of the eukaryotic cell with members of the library of ligands, and (e) measuring the sion of the reporter gene, wherein the polynucleotide cassette comprises an alternatively-spliced exon, flanked by a 5' intron and a 3' intron, and a riboswitch comprising (i) an effector region comprising a stem that includes the 5' splice site sequence of the 3' , and (ii) an r, wherein the alternatively-spliced exon comprises a stop codon that is in-frame with the reporter gene when the alternatively-spliced exon is spliced into the reporter gene mRNA.

7. The method of any one of claims 1 to 6, wherein the ligand is (a) a small molecule; or (b) a molecule produced by the eukaryotic cell selected from the group consisting of a metabolite, nucleic acid, vitamin, co-factor, lipid, monosaccharide, and second ger.

8. The method of any one of claims 1 to 7, wherein the eukaryotic cell is selected from the group consisting of a mammalian cell, an insect cell, a plant cell, and a yeast cell.

9. The method of any one of claims 1 to 8, wherein the reporter gene is selected from the group ting of (a) a fluorescent protein, luciferase, ß-galactosidase and horseradish peroxidase; or (b) a cytokine, a signaling molecule, a growth hormone, an dy, a tory RNA, a eutic protein, or a peptide.

10. The method of any one of claims 1 to 9, wherein the expression of the reporter gene is greater than 10-fold higher when the ligand specifically binds the aptamer than the reporter gene expression levels when the ligand is absent.

11. The method of any one of claims 1 to 10, wherein, the 5' and 3' introns are (a) derived from intron 2 of the human ß-globin gene; (b) each ndently from 50 to 300 nucleotides in length; or (c) each independently from 125 to 240 nucleotides in length.

12. The method of any one of claims 1 to 11, wherein the effector region stem is (a) 7 to 20 base pairs in length; or (b) 8 to 11 base pairs in length.

13. The method of any one of claims 1 to 12, wherein the alternatively-spliced exon (a) is derived from the group consisting of exon 2 of the human dihydrofolate reductase gene, mutant human Wilms tumor 1 exon 5, mouse calcium/calmodulin-dependent protein kinase II delta exon 16, and SIRT1 exon 6; (b) is the modified exon 2 from human DHFR; (c) is synthetic; or (d) has been modified by one or more of the group consisting of altering the ce of an exon splice enhancer, altering the sequence of exon splice silencer, adding an exon splice enhancer, and adding an exon splice silencer.

14. The method of claim 1, wherein the library of aptamers ses aptamers having one or more randomized nucleotides.

15. The method of claim 2, wherein the first or second primer in the two-cycle PCR ses a label selected from the group consisting of biotin, digoxigenin (DIG), bromodeoxyuridine (BrdU), fluorophore, and a chemical group used in click chemistry. '