# US 2020/0392484 A1 · Compositions and methods for identifying polynucleotides of interest

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2020/0392484 A1
- Document ID: US20200392484A1
- Status: Published application
- Kind: A1
- Application number: 16/909,979
- Filed: 2020-06-23
- Published: 2020-12-17
- Earliest claimed: 2015-06-26
- Assignees: The Regents of the University of California
- Applicants: The Regents of the University of California
- Inventors: Paul A. Feldstein; James E. Lincoln
- Web page: https://patentgrep.com/patent/US20200392484A1

## CPC

- C12N 15/1093: {General methods of preparing gene libraries, not provided for in other subgroups}
- C12N 15/1051: {Gene trapping, e.g. exon-, intron-, IRES-, signal sequence-trap cloning, trap vectors}
- C12N 15/1096: {cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR}
- C12Q 1/6869: Methods for sequencing
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

Provided are constructs and methods useful for the screening and identification of polynucleotide sequences of interest, in particular promoters, RNA stability modifying sequences and transcriptional modifying sequences.

## Claims

### Claim 1

A DNA plasmid comprising operably linked polynucleotides encoding:
(a) a mini-monomer cassette, comprising a first ribozyme cleavage site and a second ribozyme cleavage site, and comprising between the first ribozyme cleavage site and the second ribozyme cleavage site:
(i) a ribozyme catalytic core;
(ii) a multiple cloning site polynucleotide comprising plurality of restriction endonuclease cleavage sites; and
(iii) a reverse primer annealing polynucleotide located upstream or 5′ to a forward primer annealing polynucleotide; and
(b) a plasmid backbone, comprising an origin of replication.

### Claim 2 (depends on 1)

The DNA plasmid of claim 1, comprising operably linked polynucleotides encoding:
(a) a mini-monomer cassette, comprising in the 5′ to 3′ direction:
(i) a first ribozyme cleavage site;
(ii) a ribozyme catalytic core;
(iii) a multiple cloning site polynucleotide comprising plurality of restriction endonuclease cleavage sites, a reverse primer annealing polynucleotide and a forward primer annealing polynucleotide; and
(iv) a second ribozyme cleavage site;
(b) a plasmid backbone, comprising an origin of replication.

### Claim 3 (depends on 1)

The plasmid of claim 1, wherein the plasmid backbone further comprises an expression cassette encoding a selection marker, wherein the expression cassette is in the sense or antisense orientation from the ribozyme catalytic core.

### Claim 4 (depends on 3)

The plasmid of claim 3, wherein the selection marker comprises a polynucleotide encoding a gene or protein conferring antibiotic resistance, heat tolerance, fluorescence, luminescence or rescue of auxotrophic growth for specific amino acids or nucleotides.

### Claim 5 (depends on 1)

The plasmid of claim 1, wherein the ribozyme catalytic core is derived from a hairpin ribozyme, said hairpin ribozyme catalytic core derived from the negative strand self-cleavage domain of a plant virus satellite RNA selected from the group consisting of the satellite RNA of tobacco ringspot virus (sTRSV), the satellite RNA of arabis mosaic virus (sArMV), and the satellite RNA of chicory yellow mottle virus (sCYMV).

### Claim 6 (depends on 1)

The plasmid of claim 1, wherein the ribozyme catalytic core comprises a polynucleotide selected from the group of polynucleotides consisting of SEQ ID NO: 8, SEQ ID NO: 3 SEQ ID NO: 4 and SEQ ID NO: 5.

### Claim 7 (depends on 1)

The plasmid claim 1, wherein the first, second and/or third ribozyme cleavage sites are sites selected from the group of sites consisting of sites with a polynucleotide of SEQ ID NO: 6, sites with a polynucleotide of SEQ ID NO: 7 and hairpin ribozyme cleavage sites.

### Claim 8 (depends on 1)

The plasmid of claim 1, further comprising upstream of or 5′ to the mini-monomer cassette, a control promoter functional in a prokaryotic cell selected from the group of promotors consisting of a T7, a T3 and a SP6 promoters.

### Claim 9

A DNA plasmid comprising operably linked polynucleotides encoding:
(a) a mini-monomer cassette, comprising a first ribozyme cleavage site and a second ribozyme cleavage site, and comprising between the first ribozyme cleavage site and the second ribozyme cleavage site:
(i) a ribozyme catalytic core;
(ii) a reverse primer annealing polynucleotide;
(iii) a forward primer annealing polynucleotide; and
(iv) an inserted polynucleotide suspected of comprising a promoter; and
(b) a plasmid backbone, comprising an origin of replication.

### Claim 10 (depends on 9)

The plasmid of claim 9, comprising operably linked polynucleotides encoding:
(a) a mini-monomer cassette, comprising in the 5′ to 3′ direction:
(i) a first ribozyme cleavage site;
(ii) a ribozyme catalytic core;
(iii) a reverse primer annealing polynucleotide;
(iv) a forward primer annealing polynucleotide;
(v) an inserted polynucleotide suspected of comprising a promoter;
(vi) a second ribozyme cleavage site;
(b) a plasmid backbone, comprising an origin of replication.

### Claim 11 (depends on 9)

The plasmid of claim 9, wherein the second ribozyme cleavage site is capable of being transcribed by an RNA polymerase when the inserted polynucleotide comprises a functional promoter.

### Claim 12

A DNA plasmid comprising operably linked polynucleotides encoding:
(a) a test promoter, wherein the test promoter is functional in a eukaryotic cell or in a prokaryotic cell;
(b) a mini-monomer cassette, comprising:
(i) a first ribozyme cleavage site;
(ii) a ribozyme catalytic core;
(iii) a reverse primer annealing polynucleotide;
(iv) a forward primer annealing polynucleotide;
(v) an inserted polynucleotide suspected of comprising an RNA stability modifier or a transcription modifier;
(vi) a second ribozyme cleavage site;
(c) a plasmid backbone, comprising an origin of replication.

### Claim 13

A DNA plasmid comprising operably linked polynucleotides encoding:
(a) a test promoter, wherein the test promoter is functional in a eukaryotic cell or in a prokaryotic cell;
(b) a first mini-monomer cassette, comprising:
(i) a first ribozyme cleavage site;
(ii) a first ribozyme catalytic core;
(iii) a first reverse primer annealing polynucleotide;
(iv) a first forward primer annealing polynucleotide;
(v) a barcode polynucleotide
(vi) a second ribozyme cleavage site;
(c) a second mini-monomer cassette, comprising:
(i) the second ribozyme cleavage site;
(ii) a second ribozyme catalytic core;
(iii) a second reverse primer annealing polynucleotide;
(iv) a second forward primer annealing polynucleotide;
(v) an inserted polynucleotide suspected of comprising an RNA stability modifier or a transcription modifier;
(vi) a third ribozyme cleavage site;
(d) a plasmid backbone, comprising an origin of replication.

### Claim 14 (depends on 13)

The plasmid of claim 13, wherein the plasmid backbone further comprises an expression cassette encoding a selection marker encoding a gene or protein conferring antibiotic resistance, heat tolerance, fluorescence, luminescence or rescue of auxotrophic growth for specific amino acids or nucleotides.

### Claim 15 (depends on 13)

The plasmid of claim 13, wherein the polynucleotide suspected of comprising a RNA stability modifier or a transcription modifier comprises a repressor or an enhancer.

### Claim 16 (depends on 13)

The plasmid of claim 13, wherein the inserted polynucleotide suspected of comprising a RNA stability modifier or a transcription modifier is from the group consisting of a library of randomized chemically synthesized DNA sequences, a genomic DNA library and a mutagenized RNA stability modifier or a transcription modifier.

### Claim 17 (depends on 13)

The plasmid of claim 13, wherein the hairpin ribozyme catalytic core is derived from the negative strand self-cleavage domain of a plant virus satellite RNA selected from the group consisting of the satellite RNA of tobacco ringspot virus (sTRSV), the satellite RNA of arabis mosaic virus (sArMV), and the satellite RNA of chicory yellow mottle virus (sCYMV).

### Claim 18 (depends on 13)

The plasmid of claim 13, wherein the first and/or second ribozyme catalytic core comprises a polynucleotide selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 3. SEQ ID NO: 4 and SEQ ID NO: 5.

### Claim 19 (depends on 13)

The plasmid claim 13, wherein the first, second and/or third ribozyme cleavage sites are sites selected from the group of a polynucleotide of SEQ ID NO: 7, a polynucleotide of SEQ ID NO: 6 and hairpin ribozyme cleavage sites.

### Claim 20 (depends on 13)

The plasmid of claim 13, further comprising upstream of or 5′ to the mini-monomer cassette, a control promoter functional in a prokaryotic cell selected from the group of T7, T3 and SP6.