# US 11,299,769 B2 · Target reporter constructs and uses thereof

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- Number: US 11,299,769 B2
- Document ID: US11299769B2
- Status: Granted
- Kind: B2
- Application number: 15/614,958
- Filed: 2017-06-06
- Published: 2017-12-07
- Granted: 2022-04-12
- Earliest claimed: 2016-06-06
- Assignees: RedVault Biosciences, LP
- Applicants: RedVault Biosciences, LP
- Inventors: Michael L. Metzker; Christopher August Weier
- Web page: https://patentgrep.com/patent/US11299769B2

## CPC

- C12Q 1/6811: Selection methods for production or design of target specific oligonucleotides or binding molecules
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 1/6809: Methods for determination or identification of nucleic acids involving differential detection
- C12Q 1/6837: using probe arrays or probe chips (C12Q1/6874 takes precedence)
- C12Q 1/6844: Nucleic acid amplification reactions
- C12Q 1/6853: using modified primers or templates
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 1/703: {Viruses associated with AIDS}
- C12Q 1/6869: Methods for sequencing
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q 2600/178: miRNA, siRNA or ncRNA
- 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 herein are methods and compositions for the detection of target nucleic acids using target reporter constructs (TRCs) which comprise target sequences complementary to the target nucleic acid. Further provided are methods of replicating the TRCs using rolling circle replication and/or rolling circle amplification to produce replicated TRCs which can be detected using probe sequences within the replicated TRCs.

## Claims

### Claim 1

A target reporter construct (TRC) for detecting target nucleic acids, the TRC being a closed, partially single-stranded nucleic acid comprising:
(a) one or more target sequences complementary to the target nucleic acids, wherein the target sequences are toehold switches comprising a short duplex structure with an overhang;
(b) a bridge sequence forming a double-stranded portion of the TRC, wherein the bridge sequence comprises one or more stabilizing sequences for the formation of nanospheres during replication; and
(c) an accessory sequence comprising a multifunctional probe sequence.

### Claim 2 (depends on 1)

The TRC of claim 1, wherein the TRC comprises standard canonical bases, modified bases, non-natural bases, or any combination of the bases thereof.

### Claim 3 (depends on 2)

The TRC of claim 2, wherein the target nucleic acids are single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), or single-stranded RNA (ssRNA).

### Claim 4 (depends on 3)

The TRC of claim 3, wherein the ssRNA is microRNA (miRNA).

### Claim 5 (depends on 3)

The TRC of claim 3, wherein the ssRNA is a non-coding RNA (ncRNA).

### Claim 6 (depends on 1)

The TRC of claim 1, wherein the TRC comprises two target sequences separated by a bridge sequence.

### Claim 7 (depends on 1)

The TRC of claim 1, wherein the bridge sequence is between the target sequence and accessory sequence.

### Claim 8 (depends on 1)

The TRC of claim 1, wherein the TRC comprises two or more bridge sequences.

### Claim 9 (depends on 1)

The TRC of claim 1, wherein the accessory sequence further comprises at least a second probe sequence.

### Claim 10 (depends on 9)

The TRC of claim 9, wherein the first probe sequence and distinct second probe sequence form a unique multiplex probe signature.

### Claim 11 (depends on 10)

The TRC of claim 10, wherein the unique multiplex probe signature further comprises a third, fourth, or fifth probe sequence.

### Claim 12 (depends on 1)

The TRC of claim 1, wherein the multifunctional probe sequence comprises a binding sequence that facilitates localization to an oligonucleotide array.

### Claim 13 (depends on 1)

The TRC of claim 1, wherein the TRC is between 50 and 100 nucleotides in length.

### Claim 14 (depends on 1)

The TRC of claim 1, wherein the TRC is between 100 and 500 nucleotides in length.

### Claim 15 (depends on 1)

A replicated TRC comprising concatenated monomer repeats of a TRC according to claim 1, wherein the replicated TRC is produced from rolling circle replication (RCR) and rolling circle amplification (RCA).

### Claim 16 (depends on 1)

A method of detecting a target nucleic acid comprising:
(a) obtaining target reporter constructs (TRCs) according to claim 1;
(b) contacting the TRCs with a population of target nucleic acids, wherein the target nucleic acids within the population hybridize to the complementary target sequence of a target-specific TRC;
(c) performing rolling circle replication (RCR), thereby obtaining replicated TRCs;
(d) introducing at least one detectable moiety, wherein the at least one detectable moiety binds the probe sequence of the replicated TRC; and
(e) detecting the at least one detectable moiety, thereby detecting the target nucleic acid.

### Claim 17 (depends on 16)

The method of claim 16, wherein performing RCR comprises introducing a strand-displacing polymerase.

### Claim 18 (depends on 17)

The method of claim 17, wherein the strand-displacing polymerase is a DNA polymerase or RNA polymerase.

### Claim 19 (depends on 1)

The TRC of claim 1, wherein the one or more stabilizing sequence comprise a palindromic sequence.

### Claim 20 (depends on 1)

The TRC of claim 1, wherein the one or more stabilizing sequence comprise a low complexity, repetitive sequence.