Depletion probes

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.

What is claimed is: 
     
         1 . A method for depleting RNA, the method comprising:
 exposing a plurality of DNA oligos to a sample, wherein the DNA oligos form heteroduplexes with a specific subset of RNA in the sample within a predetermined range of melting temperatures but do not substantially interact with RNA that are not part of the subset; and   digesting RNA in the formed heteroduplexes.   
     
     
         2 . The method of  claim 1 , wherein a plurality of the DNA oligos form heteroduplexes along a length of RNA molecules in the subset. 
     
     
         3 . The method of  claim 2 , wherein the DNA oligos form heteroduplexes in a non-uniform distribution along RNA molecules in the subset. 
     
     
         4 . The method of  claim 1 , wherein the DNA oligos form heteroduplexes that tile along the length of members of the subset of RNA. 
     
     
         5 . The method of  claim 4 , wherein the oligos are designed to minimize spacing between adjacent heteroduplexes. 
     
     
         6 . The method of  claim 1 , wherein the predetermined range of melting temperatures is optimized to specific sequences of the DNA oligos to maximize binding to RNA in the subset. 
     
     
         7 . The method of  claim 1 , wherein the sequences of the DNA oligos are selected by:
 generating a set of candidate sequences complementary to RNA in the subset with a melting temperature within the predetermined range;   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 a set of the candidate sequences that minimizes the cost function, inter-position gaps, and overlap.   
     
     
         8 . The method of  claim 1 , wherein the subset of RNA is selected from the group consisting of ribosomal RNA, a globin transcript, and mitochondrial RNA. 
     
     
         9 . The method of  claim 1 , wherein the digesting step includes treating the sample with RNAse H. 
     
     
         10 . The method of  claim 1 , wherein the method increases a ratio of poly-A tailed RNA to non-coding RNA in the sample. 
     
     
         11 . The method of  claim 1 , wherein spacing between the heteroduplexes is mathematically random. 
     
     
         12 . The method of  claim 1 , wherein sequences of at least two of the DNA oligos are selected to hybridize to the subset of RNA at locations that overlap or abut. 
     
     
         13 . The method of  claim 1 , wherein the DNA oligos have lengths that vary from about 18 bases to about 44 bases. 
     
     
         14 . The method of  claim 1 , wherein the DNA oligos are present in the mix at non-equimolar concentrations. 
     
     
         15 . The method of  claim 9 , wherein the RNA, the DNA oligos and the Rnase H are added together in a single tube before the hybridization and digestion is carried out.