Depletion probes
AssigneeWatchmaker Genomics, Inc.
InventorsTravis J. Sanders, Martin Ranik, Brian A. Kudlow
Life of this patent
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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.
Claims12
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.
Dependencies are inferred from the claim text.