# US 2014/0154673 A1 · Probes of rna structure and methods for using the same

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- Number: US 2014/0154673 A1
- Document ID: US20140154673A1
- Status: Published application
- Kind: A1
- Application number: 13/967,161
- Filed: 2013-08-14
- Published: 2014-06-05
- Earliest claimed: 2012-08-14
- Assignees: The Board of Trustees of the Leland Stanford Junior University
- Applicants: The Board of Trustees of the Leland Stanford Junior University
- Inventors: Robert Carl Spitale; Peter Joseph Crisalli; Howard Yuan-Hao Chang; Eric Todd Kool; Jong Hwa Jung
- Web page: https://patentgrep.com/patent/US20140154673A1

## CPC

- C12Q 1/68: involving nucleic acids
- Y10T 436/143333: Saccharide [e.g., DNA, etc.]
- 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
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

Methods for obtaining structural data from RNA in a sample, and RNA probes for performing the same, are provided. Methods of reversibly modifying RNA is a sample, in vitro or in vivo, and reversible probes for performing the same, are provided. The RNA probes may be SHAPE probes that include aryl or heteroaryl acyl imidazoles. The RNA probes may be reversible probes that include an aryl or heteroaryl ring substituted with a hydroxyl-reactive group and an azido-containing group. Also provided are methods of comparing in vitro and in vivo RNA structural data. Also provided are methods of diagnosing a cellular proliferative disease condition, e.g., by probing HOTAIR RNA. Aspects of the invention further include compositions, e.g., probes and kits, etc., that find use in methods of the invention.

## Claims

### Claim 1

A method for obtaining structural data from a RNA in a sample, the method comprising:
contacting the sample with a SHAPE probe to acylate one or more unconstrained nucleotides of the RNA to produce a 2′-acylated RNA, wherein the SHAPE probe is described by formula (II):
wherein:
A is an aryl or a heteroaryl ring;
R1 is H or a lower alkyl;
R2 is H or a lower alkyl; and
analyzing the 2′-acylated RNA to obtain structural data.

### Claim 2 (depends on 1)

The method of claim 1, wherein A comprises one or more groups independently selected from a solubility-enhancing group, a binding moiety, a tag, a permeability-enhancing group, a detectable label and a bioorthogonal group.

### Claim 3 (depends on 1)

The method of claim 1, wherein the SHAPE probe is described by one of formulas (II) and (III):
wherein:
Z1, Z2, Z3, Z4 Z5, Z6 and Z7 are independently selected from O, S, CR3, N and NR3, wherein R is H or an aryl group substituent.

### Claim 4 (depends on 3)

The method of claim 3, wherein A is selected from a phenyl, a pyridyl, pyrrolyl, a furanyl, a thienyl, a thiazolyl, an imidazolyl, oxazolyl, a pyrimidinyl, a pyrazinyl and a pyridazinyl ring.

### Claim 5 (depends on 4)

The method of claim 4, wherein the SHAPE probe is described by one of the following structures:
wherein Z8 is O, S or NR4, wherein R4 is H or a lower alkyl; and
wherein the imidaolyl group is optionally substituted with a lower alkyl, a halo or nitro.

### Claim 6 (depends on 5)

The method of claim 5, wherein R1 is a lower alkyl.

### Claim 7 (depends on 6)

The method of claim 6, wherein R1 is selected from one of the following groups:
wherein X is selected from —N3, hydroxyl, protected hydroxyl, protected thiol, amino and protected amino; and
R5 is H or an alkyl group substituent.

### Claim 8 (depends on 7)

The method of claim 7, further comprising applying a stimulus to the sample to de-acylate the 2′-acylated RNA.

### Claim 9 (depends on 8)

The method of claim 8, wherein the stimulus is a photon, a deprotection reagent or an azido-reactive agent.

### Claim 10 (depends on 9)

The method of claim 9, wherein:
R1 is —CH—N3 or —CH2CH2N3; and
the stimulus is selected from an arylphosphine, an alkylphosphine, an arylalkylphosphine and a dithiol.

### Claim 11 (depends on 2)

The method of claim 2, further comprising:
contacting the sample with a modifying reagent under conditions sufficient to conjugate the reagent to a functional group of the A ring of a 2′-acylated RNA.

### Claim 12 (depends on 11)

The method of claim 11, wherein the modifying reagent is selected from a tag (e.g., a fluorophore or peptide tag) and a solid support.

### Claim 13 (depends on 1)

The method of claim 1, wherein the sample is a cell.

### Claim 14 (depends on 1)

The method of claim 1, wherein the sample is in vivo.

### Claim 15 (depends on 1)

The method of claim 1, wherein the sample is in vitro.

### Claim 16

A method for reversibly modifying RNA in a sample, the method comprising:
contacting the sample with a reversible probe to modify one or more unconstrained nucleotides of the RNA to produce a 2′-modified RNA, wherein the probe is described by formula (V):
wherein:
Y is a hydroxy-reactive group;
A is an aryl or a heteroaryl ring; and
L is a lower alkyl group;
analyzing the 2′-modified RNA to obtain structural data; and
contacting the sample with an azido-reactive reagent to reverse modification of the 2′-modified RNA.

### Claim 17 (depends on 16)

The method of claim 16, wherein Y is an active ester and the 2′-modified RNA is 2′-acylated RNA.

### Claim 18 (depends on 17)

The method of claim 17, wherein the azido-reactive reagent is selected from an arylphosphine, alkylphosphine, an arylalkylphosphine and a dithiol.

### Claim 19 (depends on 18)

The method of claim 18, wherein the azido-reactive reagent comprises a binding moiety that specifically binds to the 2′-acylated RNA adjacent to the site of 2′-acylation (e.g., oligonucleotide having a sequence that hybridizes to a region of the 2′-acylated RNA).

### Claim 20 (depends on 16)

The method of claim 16, wherein A comprises one or more groups independently selected from a solubility-enhancing group, a binding moiety a permeability-enhancing group and a functional group (e.g., for attaching a tag (e.g., a fluorophore) or a solid support).

### Claim 21 (depends on 17)

The method of claim 17, wherein the reversible probe is described by one of formulas (VI) and (VII):
wherein:
Z1, Z2, Z3, Z5, Z6 and Z7 are independently selected from O, S, CR, N and NR, wherein R is H or an aryl group substituent;
X is halo, imidazolyl, an N-hydroxylsuccinimidyl, alkoxy or an aryloxy; and
m is 1 or 2.

### Claim 22 (depends on 21)

The method of claim 21, wherein A is selected from a phenyl, a pyridyl, a pyrrolyl, a furanyl, a thienyl, a thiazolyl, imidazolyl, an oxazolyl, a pyrimidinyl, a pyrazinyl and a pyridazinyl ring.

### Claim 23 (depends on 22)

The method of claim 22, wherein the reversible probe is described by one of the following structures:
wherein:
Z8 is O, S or NR4, wherein R4 is H or a lower alkyl; and
m is 1 or 2.

### Claim 24 (depends on 23)

The method of claim 23, wherein X is imidazolyl, optionally substituted with a lower alkyl, a halo or nitro.

### Claim 25 (depends on 24)

The method of claim 24, wherein m is 1.

### Claim 26 (depends on 16)

The method of claim 16, further comprising:
contacting the sample with a modifying reagent under conditions sufficient to conjugate the reagent to a functional group of the A ring of a 2′-modified RNA.

### Claim 27 (depends on 26)

The method of claim 26, wherein the modifying reagent is selected from a detectable label (e.g., a fluorophore or peptide tag) and a solid support.

### Claim 28 (depends on 16)

The method of claim 16, wherein the sample is a cell.

### Claim 29 (depends on 28)

The method of claim 28, wherein the sample is in vivo.

### Claim 30 (depends on 16)

The method of claim 16, wherein the sample is in vitro.

### Claim 31

A method for comparing in vitro and in vivo RNA structural data, the method comprising:
contacting an in vitro sample comprising a first RNA, with a first RNA probe to acylate one or more unconstrained nucleotides of the first RNA to produce a first 2′-acylated RNA, and analyzing the first 2′-acylated RNA to obtain in vitro structural data;
contacting an in vivo sample comprising a second RNA with a second RNA probe to acylate one or more unconstrained nucleotides of the second RNA to produce a second 2′-acylated RNA in vivo, and analyzing the 2′-acylated RNA to obtain in vivo structural data;
comparing the in vitro structural data with the in vivo structural data.

### Claim 32 (depends on 31)

The method of claim 31, further comprising identifying one or more nucleotides of the RNA that are differentially acylated in vitro versus in vivo.

### Claim 33 (depends on 31)

The method of claim 31, wherein the first and second RNA probes are each independently described by formula (I):
wherein:
A is an aryl or heteroaryl ring;
R1 is H or a lower alkyl; and
Y is a hydroxyl-reactive group.

### Claim 34 (depends on 33)

The method of claim 33, wherein Y is an acyl imidazole.

### Claim 35 (depends on 33)

The method of claim 33, wherein R1 further comprises an azido group.

### Claim 36 (depends on 33)

The method of claim 33, wherein A further comprises a bioorthonal group.

### Claim at position 37

37-49. (canceled)