# US 12,601,018 B2 · Detection of SARS-CoV-2 using RNA multi-arm junction logic gates

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- Number: US 12,601,018 B2
- Document ID: US12601018B2
- Status: Granted
- Kind: B2
- Application number: 17/822,213
- Filed: 2022-08-25
- Published: 2023-05-11
- Granted: 2026-04-14
- Earliest claimed: 2021-08-25
- Assignees: Arizona Board of Regents on behalf of Arizona State University
- Applicants: Alexander Green; Duo Ma
- Inventors: Alexander Green; Duo Ma
- Web page: https://patentgrep.com/patent/US12601018B2

## CPC

- C12Q 1/701: {Specific hybridization probes}
- C12Q 1/6851: Quantitative amplification
- C12Q 1/6897: involving reporter genes operably linked to promoters
- 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

The present invention provides loop-mediated riboregulators for the detection of SARS-CoV-2. Also provided are DNA constructs encoding the loop-mediated riboregulators and methods of using the loop-mediated riboregulators to detect the presence of SARS-CoV-2 in a sample.

## Claims

### Claim 1

A loop-mediated riboregulator comprising a multi-arm junction upstream of the coding sequence of a reporter gene;
wherein the multi-arm junction comprises from 5′ to 3′: a first base stem region, at least two sensor arms, and a second base stem region, and wherein the first base stem region is at least partially complementary to the second base stem region such that the first and second base stem regions pair to form a base stem;
wherein each sensor arm comprises from 5′ to 3′: a first sensor stem region, a loop region, and a second sensor stem region, wherein the first sensor stem region is at least partially complementary to the second sensor stem region such that the first and second sensor stem regions pair to form a sensor stem, and wherein a portion of the loop region is at least partially complementary to a target RNA sequence;
wherein the multi-arm junction comprises a ribosome binding site (RBS) and start codon within one of the base stem regions or sensor stem regions such that the secondary structure of the multi-arm junction conceals the RBS and start codon in the absence of target RNA sequences, and wherein binding of one or more target RNA sequences to one or more loop regions unwinds at least a portion of the secondary structure to expose the RBS and start codon thereby enabling translation of the reporter gene; and
wherein the two or more target RNA sequences are from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

### Claim 2 (depends on 1)

The loop-mediated riboregulator of claim 1, wherein the multi-arm junction further comprises one or more hairpin reconfiguration domains positioned between the two sensor arms.

### Claim 3 (depends on 1)

The loop-mediated riboregulator of claim 1, wherein the multi-arm junction comprises at least three sensor arms.

### Claim 4 (depends on 1)

The loop-mediated riboregulator of claim 1, wherein the loop region of the at least two sensor arms is at least 15 nucleotides in length.

### Claim 5 (depends on 4)

The loop-mediated riboregulator of claim 4, wherein the loop region of the at least two sensor arms is at least 21 nucleotides in length.

### Claim 6 (depends on 1)

The loop-mediated riboregulator of claim 1, wherein the sensor stem comprises one or more bulges.

### Claim 7 (depends on 6)

The loop-mediated riboregulator of claim 6, wherein the sensor stem comprises four bulges.

### Claim 8 (depends on 1)

The loop-mediated riboregulator of claim 1, wherein one of the sensor arms has a locked configuration and the other sensor arms have an unlocked configuration.

### Claim 9 (depends on 8)

The loop-mediated riboregulator of claim 8, wherein the RBS and stop codon are positioned within the stem of the sensor arm with the locked configuration.

### Claim 10 (depends on 1)

The loop-mediated riboregulator of claim 1, wherein all of the sensor arms have an unlocked configuration.

### Claim 11 (depends on 10)

The loop-mediated riboregulator of claim 10, wherein the RBS and stop codon are positioned within the base stem.

### Claim 12 (depends on 1)

The loop-mediated riboregulator of claim 1, wherein the two or more target RNA sequences are from a SARS-CoV-2 nucleocapsid (N) gene.

### Claim 13 (depends on 12)

The loop-mediated riboregulator of claim 12, wherein each of the sensor stems comprise an RNA sequence selected from the group consisting of: SEQ ID NOs:3-6.

### Claim 14 (depends on 13)

The loop-mediated riboregulator of claim 13, wherein the multi-arm junction comprises an RNA sequence selected from SEQ ID NO:1 and SEQ ID NO:2.

### Claim 15 (depends on 1)

The loop-mediated riboregulator of claim 1, wherein the reporter gene is a resistance gene or a gene encoding a fluorescent protein.

### Claim 16 (depends on 1)

A DNA construct comprising a promoter and a sequence encoding the loop-mediated riboregulator of claim 1.

### Claim 17 (depends on 1)

A method for detecting the presence of two or more target RNA sequences from SARS-CoV-2 in a sample, the method comprising:
a) providing a sample comprising RNA;
b) contacting the sample with the loop-mediated riboregulator of claim 1;
c) detecting translation of the reporter gene;
wherein translation of the reporter gene indicates that SARS-CoV-2 is present in the sample.

### Claim 18 (depends on 17)

The method of claim 17, wherein the method is carried out in a paper-based cell-free system.

### Claim 19 (depends on 17)

The method of claim 17, wherein translation of the reporter gene produces a colorimetric readout.

### Claim 20 (depends on 17)

The method of claim 17, wherein the RNA in the sample is amplified prior to step (b).