Oligonucleotide detection method

Abstract

The invention relates to a method for the detection of oligonucleotides using anion exchange high performance liquid chromatography. Fluorescently labelled peptide nucleic acid oligomers, complementary to the oligonucleotide are hybridized to the oligonucleotides. Anion exchange high performance liquid chromatography is then performed and the hybridized moieties detected and quantitated. The invention also relates to a method for the simultaneous detection of both strands of an oligonucleotide in parallel from one sample, and a kit for use in qualitative and quantitative detection of one or two strands of an oligonucleotide.

What is claimed is: 
     
         1 . A method for detecting an oligonucleotide and its oligonucleotide metabolites, wherein the oligonucleotide is an RNA oligonucleotide selected from the group consisting of short interfering RNAs (siRNAs) and microRNAs (miRNAs), comprising the steps of
 (a) selecting a biological sample containing or suspected of containing said RNA oligonucleotide, wherein the sample is tissue lysate or plasma,   (b) combining said biological sample with a fluorescently labeled peptide nucleic acid (PNA) probe which is fully complementary to at least 10 nucleotides of said RNA oligonucleotide thereby forming hybridized moieties between said RNA oligonucleotide sequence and said PNA probe,   (c) separating said hybridized moieties from unhybridized moieties by anion exchange high performance liquid chromatography (HPLC) under conditions where unhybridized PNA probe elutes in the void volume, and   (d) quantitatively detecting said hybridized moieties by fluorescence spectroscopy.   
     
     
         2 . The method according to  claim 1 , wherein the RNA oligonucleotide is a siRNA oligonucleotide,
 wherein both strands of a siRNA oligonucleotide from one sample are detected in parallel, and   wherein said sample is contacted with a fluorescently labelled PNA probe fully complementary to a least 10 nucleotides of the sense strand of said RNA oligonucleotide and a fluorescently labelled PNA probe fully complementary to at least 10 nucleotides of the antisense strand of said RNA oligonucleotide in step (b) and then performing steps (c) to (d), wherein (i) the two PNA probes are designed so that hybridization leads to different retention times of the two single strands, or (ii) two different fluorescence labels are used.   
     
     
         3 . The method according to  claim 1 , wherein the RNA oligonucleotide is therapeutic siRNA and derivatives thereof, and
 wherein the method further comprises qualitative analysis of the in vivo metabolism of the therapeutic siRNA and derivatives thereof.   
     
     
         4 . The method according to  claim 2 , wherein the RNA oligonucleotide is therapeutic siRNA and derivatives thereof, and
 wherein the method further comprises qualitative analysis of the in vivo metabolism of the therapeutic siRNA and derivatives thereof.   
     
     
         5 . The method according to  claim 1 , wherein quantitatively detecting detects RNA oligonucleotides that are extracellular and intracellular delivered siRNA. 
     
     
         6 . The method according to  claim 2 , wherein quantitatively detecting detects RNA oligonucleotides that are extracellular and intracellular delivered siRNA. 
     
     
         7 . The method according to  claim 1 , wherein a known concentration of the RNA oligonucleotide that should be detected is added to the sample. 
     
     
         8 . The method according to  claim 2 , wherein a known concentration of the RNA oligonucleotide that should be detected is added to the sample. 
     
     
         9 . The method according to  claim 1 , wherein the PNA probe is labeled with Atto 610, Atto 425 or Atto 520. 
     
     
         10 . The method according to  claim 2 , wherein the PNA probe is labeled with Atto 610, Atto 425 or Atto 520.