# US 12,601,004 B2 · Depletion probes

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- Number: US 12,601,004 B2
- Document ID: US12601004B2
- Status: Granted
- Kind: B2
- Application number: 18/116,013
- Filed: 2023-03-01
- Published: 2023-09-07
- Granted: 2026-04-14
- Earliest claimed: 2022-03-01
- Assignees: Watchmaker Genomics, Inc.
- Applicants: Watchmaker Genomics, Inc.
- Inventors: Travis J. Sanders; Martin Ranik; Brian A. Kudlow
- Web page: https://patentgrep.com/patent/US12601004B2

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor}
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The invention provides sets of RNA depletion probes, short DNA oligos that hybridize along the length of a target RNA and mediate digestion of the target RNA by RNase H to remove super-abundant RNA molecules from a sample. Depletion probes according to the invention are designed foremost based on biochemistry and the biophysical properties of the probes so that all of the depletion probes of a set exhibit substantially uniform, consistent behavior in binding to a target RNA in a sample. Probes are principally designed to specific performance targets and biophysical properties, yielding probe sets with irregular, even apparently random, spacing along a target RNA molecule.

## Claims

### Claim 1

A method of depleting a target RNA transcript in a sample, the method comprising contacting the sample with:
a nuclease that recognizes DNA/RNA heteroduplexes and cleaves the RNA; and
a plurality of DNA oligos that are each complementary to the target RNA transcript and have minimal to no annealing to off-target transcripts, wherein:
the sequences of the DNA oligos of the plurality of DNA oligos are selected by:
(i) generating a set of candidate sequences complementary to the target RNA transcript, wherein the candidate sequences have a predicted melting temperature within a 3.0 degree variance around an initial value;
(ii) removing from the set of candidate sequences, candidate sequences that do not have minimal to no annealing to off-target transcripts; and
(iii) selecting from the set of candidate sequences, sequences of the plurality of DNA oligos using a cost function to minimize gaps between adjacent DNA/RNA heteroduplexes formed by the target RNA transcript and the plurality of DNA oligos,
wherein the DNA oligos of the plurality of DNA oligos are present in the sample at non-equimolar concentrations;
wherein the plurality of DNA oligos form DNA/RNA heteroduplexes with the target RNA transcript in the sample and gaps between the DNA/RNA heteroduplexes are 0-111 nucleotides.

### Claim 2 (depends on 1)

The method of claim 1, wherein the nuclease that recognizes DNA/RNA heteroduplexes and cleaves the RNA is an RNase H.

### Claim 3 (depends on 1)

The method of claim 1, comprising combining the sample, the nuclease that recognizes DNA/RNA heteroduplexes and cleaves the RNA, and the plurality of DNA oligos before a hybridization incubation step or a digestion incubation step.

### Claim 4 (depends on 1)

The method of claim 1, wherein the target RNA transcript comprises a ribosomal RNA, a globin transcript or mitochondrial RNA.

### Claim 5 (depends on 1)

The method of claim 1, wherein the plurality of oligos form DNA/RNA heteroduplexes with the target RNA transcript and the mode of the distribution of nucleotide distances between the DNA/RNA heteroduplexes on the target RNA transcript is zero.

### Claim 6 (depends on 1)

The method of claim 1, wherein each oligo of the plurality of DNA oligos is at least 18 bases in length.

### Claim 7 (depends on 1)

The method of claim 1, wherein each oligo of the plurality of DNA oligos is 18-44 bases in length.

### Claim 8 (depends on 1)

The method of claim 1, wherein each oligo of the plurality of oligos anneals to the target RNA transcript.

### Claim 9 (depends on 1)

The method of claim 1, further comprising selecting sequences of the oligos, wherein the selecting comprises:
generating a set of candidate sequences complementary to the target RNA transcript, wherein the candidate sequences have a predicted melting temperature within 3° C. of each other;
assigning a cost function to each of the candidate sequences based at least in part on a match score to reference off-target RNAs; and
selecting, from the candidate sequences, sequences of the oligos of the plurality of DNA oligos that minimize the cost function, inter-position gaps, and overlap.

### Claim 10 (depends on 1)

The method of claim 1 comprising depleting an additional target RNA transcript in the sample, the method further comprising contacting the sample with an additional plurality of DNA oligos that are each complementary to and specific to the additional target RNA transcript, wherein:
(i) each oligo of the plurality of DNA oligos and each oligo of the additional plurality of DNA oligos has a melting temperature within 3° C. of each other; and
(ii) the additional plurality of oligos comprises oligos that are complementary to different regions of the additional target RNA transcript, wherein distribution of nucleotide distances between the different regions is non-uniform.

### Claim 11 (depends on 3)

The method of claim 3, wherein the target RNA transcript and the additional target RNA transcript are selected from a ribosomal RNA, a globin transcript, and/or a mitochondrial RNA.

### Claim 12 (depends on 3)

The method of claim 3, wherein each oligo of the plurality of DNA oligos is 18-44 bases in length.