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.

Claims

1. A method for detecting a target therapeutic RNA oligonucleotide having a pre-defined sequence and RNA oligonucleotide metabolites of said target therapeutic RNA oligonucleotide, comprising the steps of: (a) preparing a sample containing or suspected of containing said target therapeutic RNA oligonucleotide having said pre-defined sequence and said RNA oligonucleotide metabolites of said target therapeutic RNA oligonucleotide, wherein said target therapeutic RNA oligonucleotide has a length of 10 nucleotides up to 29 nucleotides, and wherein said RNA oligonucleotide metabolites are said target therapeutic RNA oligonucleotide from which 1 or more nucleotides have been deleted from the 3- and/or the 5-end, and/or said RNA oligonucleotide metabolites are said target therapeutic RNA oligonucleotide comprising phosphorylated 3- or 5-ends, and wherein said sample is an extracellular or intracellular sample, (b) forming a hybridization mixture by contacting the sample with a fluorescently labeled peptide nucleic acid (PNA) probe, (c) hybridizing the PNA probe to said target therapeutic RNA oligonucleotide having said pre-defined sequence and hybridizing the PNA probe to said RNA oligonucleotide metabolites of said target therapeutic RNA oligonucleotide, wherein said PNA probe and said target therapeutic RNA oligonucleotide having said pre-defined sequence are fully complementary over at least 10 nucleotides of said target therapeutic RNA oligonucleotide having the pre-defined sequence, (d) separating hybridized moieties formed between said PNA probe and said target therapeutic RNA oligonucleotide having said pre-defined sequence, and hybridized moieties formed between PNA probe and said RNA oligonucleotide metabolites of said target therapeutic RNA oligonucleotide, from unhybridized moieties by anion exchange high performance liquid chromatography (HPLC), wherein signals associated with said hybridized moieties formed between said PNA probe and said RNA oligonucleotide metabolites of said target therapeutic RNA oligonucleotide are separated from a signal associated with hybridized moieties formed between said PNA probe and said target therapeutic RNA oligonucleotide, and (e) detecting quantitatively in a single fluorescence spectroscopy measurement said hybridized moieties formed between said PNA probe and said target therapeutic RNA oligonucleotide having said pre-defined sequence and hybridized moieties formed between said PNA probe and said RNA oligonucleotide metabolites of said target therapeutic RNA oligonucleotide.

2. The method according to claim 1 for detecting both strands of a target therapeutic RNA oligonucleotide duplex having a pre-defined sequence and RNA oligonucleotide metabolites of said target therapeutic RNA oligonucleotide in parallel from one sample, comprising the addition of a second fluorescently labeled PNA probe subsequent to step (b) and then performing steps (c) to (e), wherein said first and said second fluorescently labeled PNA probe are designed as such that (i) hybridization leads to different retention times of the two strands of the target therapeutic RNA oligonucleotide duplex and its corresponding RNA oligonucleotide metabolites of said target therapeutic RNA nucleotide duplex, or (ii) two different fluorescence labels are used.

3. The method according to claim 1, wherein said target therapeutic RNA oligonucleotide having said pre-defined sequence is a siRNA, an antisense RNA or a microRNA.

4. The method according to claim 1, wherein a known concentration of a calibration RNA oligonucleotide detectable by said PNA is added to the sample.

5. The method according to claim 1, wherein the PNA probe is labeled with Atto 610, Atto 425 or Atto 520.

6. The method according to claim 1, wherein the sample is a tissue sample, a plasma sample, or an in vitro cell sample.

7. The method of claim 1, further comprising quantifying the amount of the target therapeutic RNA oligonucleotide having said pre-defined sequence and RNA oligonucleotide metabolites of said target therapeutic RNA oligonucleotide by comparing the detected hybridized moieties to a calibration curve generated from a dilution series of the target therapeutic RNA oligonucleotide having said pre-defined sequence and the PNA probe in buffer.

8. The method of claim 1, wherein the sample is a plasma, serum or tissue sample and the preparing in (a) comprises lysing cells in the sample.

9. The method of claim 1, wherein the hybridizing in (c) comprises mixing the target therapeutic RNA oligonucleotide having a pre-defined sequence with the PNA probe and incubating at 95 C. to form the hybridized moieties.

10. The method of claim 1, wherein the preparing in (a) comprises treating the sample with proteinase K.

11. The method of claim 1, wherein the separating in (c) is conducted under native anion exchange HPLC conditions at 50 C. with NaClO.sub.4 as an eluent salt.

12. The method of claim 1, wherein the separating in (c) further comprises non-hybridized PNA probe eluting in the void volume of the HPLC.

Description

SHORT DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows a chromatogram for the detection of one strand of the siRNA.

(2) FIG. 2 shows a simultaneous analysis of both strands using two PNA probes with the same dye.

(3) FIG. 3 shows calibration curves for the simultaneous analysis of both strands using two PNA probes with the same dye.

(4) FIG. 4A-4B shows simultaneous analysis of both strands using two PNA probes with different fluorescence labels, detection with two fluorescence detectors.

(5) FIG. 5A-5G shows separation of drug metabolites.

(6) FIG. 6A-6C shows a chromatogram for the detection of miRNAs.

(7) FIG. 7A-7B shows a chromatogram for detection of spiegelmer in lung tissue.

(8) FIG. 8A-8C shows retention time shift by 3-elongated as-strand sequences.

(9) FIG. 9A-9B shows increase in sensitivity by higher tissue loading.

EXAMPLES

Example 1: Detection of GFP-siRNA by PNA-Probe HPLC

(10) This material and method section describes the assay procedure how to determine a GFP-siRNA from biological samples. Additionally this procedure can be also used with small variations for all other oligonucleotides that can form Watson-crick base pairs. The procedure allows the determination of only one strand in the case of single- and double stranded oligonucleotides and the quantification of both strands in parallel from double stranded oligonucleotides, e.g. siRNA. The dye-probe is an fluorescently labeled PNA (Peptide Nucleic Acid) strand that is fully complementary to at least 10 or more nucleotides of the oligonucleotide that should be quantified (complementary is defined as perfect Watson-Crick base pairing).

(11) Plasma, serum or tissue samples are shipped on dry ice and stored at 80 C. until used. Prior to the analysis plasma samples are thawed on ice and processed by a proteinase K treatment in Epicentre Cell and Tissue Lysis Solution at 65 C. for 25 min. For the proteinase K treatment 30 A plasma are mixed with 30 A Epicentre Cell and Tissue Lysis Solution, 4 A proteinase K solution and 36 l H.sub.2O to a final volume of 100 l.

(12) Tissues samples are pulverized in frozen state and up to 100 mg frozen powder were suspended in 1 mL Epicentre Cell and Tissue Lysis Solution, treated with an ultrasonic stick and subsequently lysed with a proteinase K treatment at 65 C. All proteinase K treated samples are further diluted with Epicentre Cell and Tissue Lysis Solution before employed in the HPLC sample preparation step.

(13) After the proteinase K treatment 20 l of a 3M KCl solution is added to 200 l of the plasma or tissue samples to precipitate the SDS. Subsequently the samples are centrifuged for 15 min and the supernatant is further used for siRNA determination.

(14) For hybridization, 100 l of the diluted supernatant containing between 0.5 and 250 fmol siRNA, is mixed in 96-PCR well plates with 5 l of a 1 M Atto610-PNA-probe solution targeting the antisense strand. Hybridization buffer is added to a final volume of 200 l (to 190 l if the sense strand of the siRNA duplex should be detected also). The plate is sealed and incubated at 95 C. for 15 min in a PCR instrument.

(15) The temperature of the PCR instrument is lowered to 50 C. If the sense strand of the siRNA duplex should be detected 10 l of a 1 M Atto425-PNA-probe (or of the Atto610-PNA-probe) targeting the sense strand is added to each well for a final volume of 200 l. After shaking for additional 15 min at 50 C. are cooled to room temperature and the samples are put into the HPLC autosampler.

(16) Calibration curves are generated from a siRNA dilution series under identical conditions. A representative chromatogram of the calibration curve used in the analysis of both strands of an oligonucleotide is provided in FIG. 3.

(17) TABLE-US-00001 TABLE1 SequencesofPNA-Probesusedfordetectionofa siRNAtargetingGFP GFP-siRNAprobeset Seq.Id.No.1 5-Atto425-(OO)-TCGTGCTGCTTCATG-3 Sense Seq.Id.No.2 5-Atto610-(OO)-TCGTGCTGCTTCATG-3 Sense Seq.Id.No.3 5-Atto610-(OO)-ACATGAAGCAGCACG-3 Antisense

(18) HPLC Analysis with Fluorescence Detection of the Probe/Antisense Strand Duplex

(19) 100 l of each hybridized sample () are injected into the HPLC system connected to a Dionex RF2000 fluorescence detector. For detection of both siRNA strands with the two different fluorescence dyes a second Dionex RF2000 fluorescence detector is used connected in a row after the first detector. The chromatography is conducted at 50 C. under native conditions with NaClO.sub.4 as eluent salt on a Dionex DNA Pac PA100 column.

(20) A typical chromatogram for the detection of one strand is shown in FIG. 1, a typical chromatogram for the simultaneous analysis of both strands using two PNA probes with the same dye is provided in FIG. 2. A representative chromatogram of the calibration curve used in the analysis of both strands of an oligonucleotide is provided in FIG. 3. In FIG. 4 a typical chromatogram of the simultaneous analysis of both strands using two PNA probes with different fluorescence labels and their detection with two fluorescence detectors is shown.

(21) HPLC-Conditions: Column: Dionex DNAPac PA100 (4250 mm analytical column) Temp.: 50 C. Flow: 1 ml/min Injection: 100 Detection: Excitation: 612 nm; Emission: 642 nm (first detector) Excitation: 436 nm; Emission: 484 nm (second detector if needed)

(22) TABLE-US-00002 TABLE 2 HPLC Gradient Table Time % A % B 1.00 min 91 9 1.00 min 91 9 9.0 min 80 20 9.5 min 0 100 12.5 min 0 100 13.0 min 91 9 16.0 min 91 9

(23) The concentrations of the GFP-siRNA in plasma and tissue samples are determined using ion-exchange HPLC to separate the analytes and quantify the area under the peak with fluorescence detection. Under the native IEX-HPLC conditions used, interfering matrix compounds as well as excess of the fluorescence labeled probes elute in the void volume of the column. Non-specific signals from hybridization of the fluorescence labeled probes with matrix RNA/DNA are shifted to higher retention times allowing for good resolution of signal with little co-eluting background. The specific signals generated by the duplexes consisting of fluorescent labeled probes and the corresponding intact siRNA strand typically elutes between 5 to 7 min.

(24) Quantitation is performed based on an external calibration curve generated from a standard siRNA dilution series (from 0.5 to 250 fmol siRNA) which is hybridized and analyzed as described above. The linear range of this assay is from 0.5 to 250 fmol siRNA on the column with an LLOQ of 0.6 ng siRNA in 1 mL plasma and 5 ng siRNA in tissue.

(25) Reagents: 50 M Standard GFP-siRNA stock solution (in house prep) Hybridization Buffer: 50 mM TRIS-Cl; 10% ACN (in house prep.) Proteinase K (20 mg/ml): Peqlab No. 04-1075; Lot: 11024 Lysis buffer: Epicentre Cell and tissue lysis solution (# MTC096H) MilliQ-water: 18.2 M PNA-Probes: see Table 1 KCl: 3M solution in H.sub.2O (in house prep) HPLC-System A for fluorescence detection: HPLC Eluent A: 25 mM TRIS-HCl; 1 mM EDTA; 50% ACN; pH=8 HPLC Eluent B: 800 mM NaClO.sub.4 in A

(26) Material: Ultrasonic stick, Bandelin Sonoplus (Berlin), HD 2070 MS72 with UW 2070 1.5 ml Eppendorf tubes Eppendorf twin.tec PCR plate 96 (#951020389) Eppendorf Mastercycler gradient Ultra Clear cap-Stripes, Peqlab (#82-0866-A) Dionex Ultimate 3000 HPLC: Solvent Rack Dual Pump Ultimate 3000 Autosampler Ultimate 3000 Column Oven Ultimate 3000 with 10 port switch valve UV-Detector VWD 3000 Fluorescence-Detector RF2000
Alternatively, the following HPLC conditions were used for the detection of oligonucleotides, especially for detection of miRNAs and siRNAs: Column: Dionex DNA Pac PA100 (2504 mm) Temperature: 50 C. Eluent A: 10 mM Sodiumphosphate; 100 mM NaCl; 5% ACN Eluent B: 10 mM Sodiumphosphate; 1M NaCl; 5% ACN Eluent C: 90% ACN

(27) TABLE-US-00003 TABLE 3 HPLC Gradient Table - alternative protocol (Standard conditions for detection of miRNAs and siRNAs) Time Flow Eluent A Eluent B Eluent C [min] [mL/min] [%] [%] [%] 0.00 1.00 40 20 37 1.00 1.00 40 20 37 10.00 1.00 8 55 37 10.50 1.00 0 90 10 13.50 1.00 0 90 10 14.00 1.00 40 20 37 17.00 1.00 40 20 37

Example 2: Automated 96-Well Plate Preparation

(28) This section describes a new sample preparation protocol making use of microtiter plates. Therein, manual handling steps are reduced to a minimum to improve the reproducibility of the assay. All components of the mixture including hybridization buffer, dye-probe and the siRNA spike are added by a pipetting robot to a 96-well plate. Also the preceding SDS precipitation of the samples can be performed in a microtiter plated based setup.

(29) With his procedure it is possible to prepare 96-well plates on stock for a defined oligonucleotide, wherein only the sample solution has to be added. Accordingly, this ready-to-use preparation works like a kit and can be used for quick high-throughput analysis of samples containing or suspected of containing defined oligonucleotides.

(30) Plate Preparation

(31) In a 96 well microtiter plate the wells form a rectangular grid of 8 rows (labeled A through H) and 12 columns (labeled 1 through 12). For an automated 96 well plate preparation, a mastermix is prepared manually according to table 4 and 100 l are added to each well of the plate by a pipetting robot. To wells in row 1-10, 50 l water is added. Row 1-9 serve for sample analysis, row 10 serves as control for the 1 fmol spike and rows 1-12 for the calibration curves. To wells 11-12, 50 l medium and 50 l of a siRNA dilution are added. The siRNA dilutions are prepared by the pipetting robot starting with a 100 nM siRNA solution and are listed below. This 96-well plate is further referred to as prepared plate.

(32) TABLE-US-00004 TABLE 4 Mastermix for plate preparation Mastermix substance per vial (=500) Water 31 l 15500 l PNA Probe 5 l 2500 l l pmol/l 1 fmol- 4 l 2000 ll 5 siRNA-Spike: 0.5 fmol/l Acetonitril 20 l 10000 l 1M Tris pH 8.0 40 l 20000 l

(33) siRNA Dilutions for Calibration Curves 20 nM (500 fmol) 10 nM (250 fmol) 4 nM (100 fmol) 2 nM (50 fmol) 1 nM (25 fmol) 0.4 nM (10 fmol) 0.2 nM (5 fmol) 0.1 nM (2.5 fmol) 0.02 nM (0.5 fmol) 0.01 nM (0.25 fmol)

(34) Addition of Samples to Prepared Plate/SDS Precipitation Step

(35) 100 l aliquots of samples are pipetted to wells into all rows (A-H) of columns 1 to 9 of a precooled 96 well microtiter plate, and to these wells 10 l 3M KCl are added by a pipetting robot. After 15 minutes of centrifugation at 3800 U/min and 4 C., 50 l of the supernatant are transferred by the pipetting robot to the according columns of a prepared plate.

(36) For the control, lysis buffer or medium is precipitated with 3M KCl and 50 l of the supernatant added to column 10-12 of the prepared plate. 100 l of each well are injected onto aIEX-HPLC.

Example 3: Separation of Drug Metabolites

(37) For separation of different drug metabolites purified 3 end (3 n-2, 3 n-4, 3 n-5, 3 n-6) and 5 end (5 n-1, 5n-2, 5n-3) metabolites of the as strand of GFP-siRNA, from which 1 to 6 nucleotides were deleted from the 3 or 5 end, respectively, were analysed according to the assay procedure described in Example 1. Metabolites are given in table 5, a typical chromatogram for separation of metabolites is given in FIG. 5.

(38) TABLE-US-00005 TABLE5 RepresentativemetabolitesofGFP-siRNA GFP-siRNA Name Sequence Seq.Id Sense GFP-siRNA-s-strand 5-CCACAUGAAGCAGCACGACUU-3 4 Antisense GFP-siRNA-as-strand 5-AAGUCGUGCUGCUUCAUGUGGUC-3 5 GFP-siRNA-as-strand-5-(n 1) 5-AGUCGUGCUGCUUCAUGUGGUC-3 6 GFP-siRNA-as-strand-5-(n 2) 5-GUCGUGCUGCUUCAUGUGGUC-3 7 GFP-siRNA-as-strand-5-(n 3) 5-UCGUGCUGCUUCAUGUGGUC-3 8 GFP-siRNA-as-strand-3-(n 2) 5-AAGUCGUGCUGCUUCAUGUGG-3 9 GFP-siRNA-as-strand-3-(n 4) 5-AAGUCGUGCUGCUUCAUGU-3 10 GFP-siRNA-as-strand-3-(n 5) 5-AAGUCGUGCUGCUUCAUG-3 11 GFP-siRNA-as-strand-3-(n 6) 5-AAGUCGUGCUGCUUCAU-3 12

Example 4: Detection of miRNA

(39) The assay was used under standard conditions to evaluate the possibility to detect miRNA from tissue lysates. As an example the mouse liver specific miRNA-122 was detected from mouse tissue lysate (positive control), jejunum (negative control) and from lysate spiked with synthetically generated miRNA-122 strands (Lagos-Quintana, et al. Current Biology, Vol. 12, 735-739). From literature it is known, that in liver of mice three different types of miRNA-122 sequences are expressed:

(40) TABLE-US-00006 miRNA-122a: 5-UGGAGUGUGACAAUGGUGUUUG-3 (Seq.ID.No.13) miRNA-122b: 5-UGGAGUGUGACAAUGGUGUUUGU-3 (Seq.ID.No.14) miRNA-122c: 5-UGGAGUGUGACAAUGGUGUUUGA-3 (Seq.ID.No.15)

(41) All synthetic standards were synthesized as 5-OH and as 5-Phosphate sequence. As the three species showed small variations at the 3-end the PNA-Probe was designed in way that it fully matches with all three miRNA-122 sequences, starting at the third base of the 5-end of the miRNA-122 with 17 bases in length:

(42) TABLE-US-00007 PNA-Probe: (Seq.ID.No.16) 5-Atto425-OO-AACACCATTGTCACACT-3

(43) HPLC was performer with the conditions as described in the alternative protocol detailed in example 1 and shown in table 3. HPLC-traces generated from mouse lung lysate (miRNA122 negative tissue) spiked with synthetically generated miRNA-122 showed three separated peaks. The retention time of this peaks fully match with signals, that were found in lysates from liver (1 mg liver injected). The quantitation of the total peak area and calculation of the total miRNA-122 concentration in liver lead to approximately 35 ng/g. The miRNA-122 negative control from jejunum or lung tissue samples showed no signal for miRNA-122 as expected (FIG. 6).

Example 5: Detection of Spiegelmer-DNA (L-DNA) with and without Pegylation

(44) Spiegelmers are aptamer molecules with non-natural L-ribose (L-RNA) or L-deoxyribose (L-DNA) sugar backbone that show no Watson-Crick base pair interaction with the natural D-oligonucleotides. As PNA is a non-chiral mimic of oligonucleotides with Watson-Crick base pair properties it was expected, that the PNA-probes can also be used to detect this non-natural oligonucleotide species. To increase the circulation half life of spiegelmers or aptamers this molecules are often pegylated with branched 40 kDa PEG, that usually hampers the analysis of this complex molecules.

(45) As a proof of concept for the detection of this therapeutically interesting molecule class a pegylated and a non-pegylated version of the following L-DNA sequence were synthesized and analysed with the here described assay after orotrachael administration in mice:

(46) Non-PEG-Spiegelmer:

(47) TABLE-US-00008 (Seq.ID.No.17) (NH2C6)-CCAGCCACCTACTCCACCAGTGCCAGGACTGCTTGAGGGT
PEG-Spiegelmer:

(48) TABLE-US-00009 (Seq.ID.No.18) PEG(40kDa)-(NHC6)-CAGCCACCTACTCCACCAGTGCCAGGACTG CTTGAGGGT

(49) The following 17 mer-PNA-Probe was used for hybridization and detection of the spiegelmer from plasma, lung, liver and kidney samples:

(50) TABLE-US-00010 (Seq.ID.No.19) 5-Atto425-OO-GTCCTGGCACTGGTGGA-3

(51) Gradient conditions were adjusted to the longer oligonucleotide sequences compared with to the siRNA strands to elute the spiegelmer-PNA-duplex within the gradient of the HPLC method.

(52) The following HPLC conditions were applied: Column: Dionex DNA Pac PA100 (2504 mm) Temperature: 50 C. Eluent A: 10 mM Sodiumphosphate; 100 mM NaCl; 5% CAN Eluent B: 10 mM Sodiumphosphate; 1M NaCl; 5% ACN Eluent C: 90% ACN

(53) TABLE-US-00011 TABLE 6 HPLC gradient conditions for pegylated spiegelmer Time Flow Eluent A Eluent B Eluent C [min] [mL/min] [%] [%] [%] 0.00 1.00 40 20 40 1.00 1.00 40 20 40 10.00 1.00 5 55 40 10.50 1.00 0 60 40 13.50 1.00 0 60 40 14.00 1.00 40 20 40 17.00 1.00 40 20 40

(54) TABLE-US-00012 TABLE 7 HPLC gradient conditions for Spiegelmer Time Flow Eluent A Eluent B Eluent C [min] [mL/min] [%] [%] [%] 0.00 1.00 35 25 40 1.00 1.00 35 25 40 10.00 1.00 0 60 40 10.50 1.00 0 60 40 13.50 1.00 0 60 40 14.00 1.00 35 25 40 17.00 1.00 35 25 40

(55) Sensitivity of the method was a little bit compromised for the pegylated Spiegelmer due to peak broadening induced by the polydisperity of the 40 kDa PEG-moiety. Lower limit of detection was increased to 1 fmol L-DNA on column. Resolution of shorter impurities was not tested, but expected to be lower compared to the shorter siRNA or miRNA strands.

(56) Sample preparation was done according to the standard protocol. The Spiegelmers could be easily detected by this procedure from plasma and all tissue tested, as a sharp single peaks with nearly no biological background interference as shown in FIG. 7.

Example 6: Detection of siRNA from In Vitro Transfection Experiments

(57) Detection of unlabeled siRNA from in vitro cell culture experiments was limited by the fact of the high sensitivity needed and therefore only approaches with amplification step like PCR were successful for unmodified molecules.

(58) The PNA-HPLC assay sensitivity was in range to measure siRNA from cell culture experiments. An 19 base pair siRNA with 2 nt overhang at the 3-end of both strands was used for transfection of primary hepatocytes at a 30 nM siRNA concentration. Various versions of this duplex with identical sequences, only differing at their 5-end of the antisense strand were transfected. After transfection the cells were washed with PBS and then lysed by a proteinase K treatment with a concentration of 2500 cells per uL lysate.

(59) The cell culture lysate was used for the PNA-HPLC assay procedure and 50000 cells per HPLC run were injected onto the column after hybridization with the complementary antisense strand PNA-probe. Under this assay conditions the intact as-strand and also the 5-phosphorylated species of the antisense strand could be detected down to approximately 8000 siRNA copies per cell (data not shown).

Example 7: Use of Internal Standards for Normalization (Higher Accuracy)

(60) To further increase the accuracy of the method, especially when used in a GxP environment it is maybe necessary to implement an internal standard for normalization. As a proof of concept a 21 mer RNA-strand was elongated with 3 up to 8 desoxy-T nucleotides at its 3-end. This normalization standards, together with the 21 mer and its 5-phosphorylated species were spiked into plasma and then analysed under standard assay and HPLC conditions (see example 1, especially the alternative protocol for HPLC and table 3) for siRNAs.

(61) All elongated standards eluted fully baseline resolved from the 21 mer as well as from the 5-phosphorylated 21 mer with higher retention times. Some peak interferences were observed with the 3-dT-nucleotide elongated sequence and the 5-phosphorylated 21 mer, as some synthesis impurities of the elongated strand co-eluted with the 5-phosphorylated 21 mer.

(62) The example shown here is a chromatogram overlay of samples containing the as-strand and the 5-phosphorylated as-strand mixed with the 3-elongated as-strand with 3, 4 and 5 desoxythymidine nucleotides at its 5-end (see FIG. 8). The following sequences were used for the example chromatograms (Letters in capitals represent RNA nucleotides, lower case letters c, g, a and u represent 2 O-methyl-modified nucleotides, s represents phosphorothioate and dT deoxythymidine):

(63) TABLE-US-00013 as-strand: (Seq.ID.No.20) 5-UCGAAGuACUcAGCGuAAGdTsdT-3 as-strand-5-PO.sub.4: (Seq.ID.No.21) 5-pUCGAAGuACUcAGCGuAAGdTsdT-3 as-strand-(dT).sub.n: (Seq.ID.No.22, Seq.ID.No.23, Seq.ID.No.24) 5-UCGAAGuACUcAGCGuAAGdTdT-(dT).sub.n-3 withn= 3,4,5 PNA-Probe: (Seq.ID.No.25) 5-Atto425-OO-CTTACGCTGAGTACTTC-3

(64) TABLE-US-00014 TABLE 8 Peak resolution calculated according to the USP Retention Time Resolution to Sequence [min] 5-PO4 (USP) as-strand 6.23 1.70 as-strand - 5-PO4 6.54 as-strand + 3x 3-dT 6.68 <1 as-strand + 4x 3-dT 6.88 2.25 as-strand + 5x 3-dT 7.04 2.75

(65) With this experiment it was also proven, that under standard miRNA and siRNA assay conditions baseline resolution can be achieved for oligonucleotides up to 29 mers, that differ only by one nucleotide in length.

Example 8: Increase of Assay Sensitivity in Tissues

(66) The sensitivity of the assay as described above was restricted to 2 ng siRNA per g tissue. This limitation was given by the fact that the maximal loaded tissue amount on the column was 2-3 mg per injection, as the baseline noise increased at higher tissue loadings. Switching from the Atto610 dye to the Atto425 dye allows much higher column loadings up to 11 mg without loss of signal sensitivity and chromatographic resolution power. The absolute amount of siRNA at the LOD is still 250 amol oligonucleotide on column. This lead to a lower limit of detection in respect to siRNA tissue concentration of 400 pg/g (FIG. 9).

(67) In table 9 below a comparison between two chromatographic runs of the same tissue sample, but two separate tissue preparations is shown. In the upper chromatogram 3 mg liver was loaded onto the HPLC column, in the lower chromatogram 11.2 mg was loaded.

(68) Although the results were generated from two different tissue lysate, the calculated tissue siRNA and metabolite concentrations show only minor variation:

(69) TABLE-US-00015 TABLE 9 Signal/Noise-Values of identical tissue sample; different tissue amounts loaded on HPLC column 3 mg Tissue 11 mg Tissue loaded on HPLC loaded on HPLC Ret. Tissue Ret. Tissue Time Conc. Time Conc. Tissue Conc. Peak No. min ng/g S/N min ng/g S/N Delta [ng/g] 1 4.67 17.1 67 4.59 13.5 26.8 3.5 2 5.10 4.8 18 5.02 4.3 8.3 0.6 3 5.55 20.6 87 5.47 17.5 22.8 3.1 4 5.86 19.4 81 5.80 16.3 17.9 3.0 5 6.16 10.4 44 6.09 9.0 8.7 1.3 6 6.47 14.3 61 6.42 12.5 15.7 1.8 7 6.88 7.7 33 6.83 6.8 9.8 0.9