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Pub. No.: US 2008/0050721 A1 Sina Stab 7 Sense Strand Standard Curve

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Pub. No.: US 2008/0050721 A1 Sina Stab 7 Sense Strand Standard Curve US 20080050721A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2008/0050721 A1 Radka et al. (43) Pub. Date: Feb. 28, 2008 (54) DETECTION AND QUANTITATION OF Publication Classification NUCLEC ACID MOLECULES IN (51) Int. Cl. BOLOGICAL SAMPLES CI2O I/68 (2006.01) (75) Inventors: Susan Radka, Arvada, CO (US); Zinnen Shawn, Denver, CO (US); (52) U.S. Cl. .............................................. 435/6: 436/94 Vasant Jadhav, Longmont, CO (US); James McSwiggen, Boulder, CO (US); Narendra Vaish, (57) ABSTRACT Denver, CO (US) The present invention concerns processes for the detection Correspondence Address: and quantitation of nucleic acid molecules, polynucleotides, MCDONNELL, BOEHNEN, HULBERT AND and/or oligonucleotides in a sample using hybridizationde BERGHOFF, LLP tection assays, antibody-mediated recognition assays, 300 SOUTH WACKER DRIVE, SUITE 3100 nucleic acid sensor molecules, chromatographic assays, and/ CHICAGO, IL 60606 or electrophoresis assays. The present invention specifically concerns processes for the detection and quantitation of (73) Assignee: Sirna Therapeutics, Inc., Boulder, double stranded nucleic acid molecules, polynucleotides, CO (US) and/or oligonucleotides in a sample using hybridization detection assays. The nucleic acid molecules, polynucle (21) Appl. No.: 10/568,134 otides, and/or oligonucleotides can include molecules that mediate RNA interference, such as short interfering nucleic (22) PCT Filed: Aug. 17, 2004 acid (siNA), short interfering RNA (siRNA), double stranded RNA (dsRNA), micro-RNA (miRNA), and short (86). PCT No.: PCT/USO4/26857 hairpin RNA (shRNA) molecules. The nucleic acid mol ecules, polynucleotides, and/or oligonucleotides can include S 371 (c)(1), nucleic acid aptamers, enzymatic nucleic acid molecules, (2), (4) Date: Apr. 12, 2007 decoys, antisense, 2.5'-oligoadenylate molecules, triplex forming oligonucleotides or any other nucleic acid molecule Related U.S. Application Data of interest. The present invention also concerns kits that (60) Provisional application No. 60/497,222, filed on Aug. allow for the detection and quantitation of nucleic acid 22, 2003, provisional application No. 60/543,784, molecules, polynucleotides, and/or oligonucleotides in a filed on Feb. 11, 2004. sample. siNA Stab 7 Sense Strand Standard Curve 3.5 -0- SS 7 3 --Duplex 7 2 1 S O. 1000 fmols/ml Patent Application Publication US 2008/0050721 A1 -¿OSETdWVXH, sw??s?sNõ?v?LIÑyno(TvNouºo) LEICT3)NISSEJOOÀldEITCHWV/S/NOILOE CJE|\/|LNERHEI-H-HIG] e?duueseu?VNISJO STTEOCIETOOdè?O uo?e??uenopueuol?.09.1907:V?.?un61-I Panssil Patent Application Publication Feb. 28, 2008 Sheet 2 of 27 US 2008/0050721 A1 pe?eqelºsep?XOued SOLIH??ue EKONEITUDESVNIS Messyuo?ez?p?uqÁHVN?s:g?aun61-I @@@ @- LdW/O?oºEICIILOETOTINOS)ITOERIT) EICIILOETOTINO5)ITONOILOELEIC] Patent Application Publication Feb. 28, 2008 Sheet 4 of 27 US 2008/0050721 A1 O) N O S O 3 N CM) e N 3 S (/) O s 2 (M) H N 9 Q CM) S. 2 (V) V i Patent Application Publication Feb. 28, 2008 Sheet 5 of 27 US 2008/0050721 A1 SN O S S S CM) 3 O s S O (M) 3 ; E S R O S E S. O OO V -Q CM) S 9 UUUOG7 e OO Patent Application Publication Feb. 28, 2008 Sheet 6 of 27 US 2008/0050721 A1 O) O O S S. O O S E. O Y. S. s O 2 s E SS rais. N CDSS S. 9 C(/) Q S 2 k. o H w S (51. O2Of vmO CMO S C/D D S. 5 (6 dB 5 : , (V) s s FS (5 9 i UUUOG7 le OO Patent Application Publication Feb. 28, 2008 Sheet 7 of 27 US 2008/0050721 A1 UUUOC7 e OO Patent Application Publication Feb. 28, 2008 Sheet 8 of 27 US 2008/0050721 A1 e O) O e S s w S to ge SidSS O. 3 S E co 5 NS g O vs.S e to 5 SS is ... CyS v UUUOG7 le OO Patent Application Publication Feb. 28, 2008 Sheet 9 of 27 US 2008/0050721 A1 s O O N O 8 5 ver O S. S S S is V (VS (M) OO S o E SS 9 : O O O M &N S C C 9 33 UUUO97)e OO Patent Application Publication xejdna.Zqe?SVN?s:O8ºun61-I ajduesJouquo.OÁ}I?ent) Patent Application Publication Feb. 28, 2008 Sheet 11 of 27 US 2008/0050721 A1 Sa (s 2 O Q 3 SSS SS SN S.S 5 SS S SS N N 8 S. 2 5 9 se - Q S N& is SS V S.SS Q in CM) UuuOg7 e OO S. 2 (/) Patent Application Publication Feb. 28, 2008 Sheet 12 of 27 US 2008/0050721 A1 Ie?uegodgouo?yoayad:gvaun61-I Messy uogez?puqÁHe?aseliloqejaw8qe?SVN?s uuOgeLo OO Patent Application Publication Feb. 28, 2008 Sheet 13 of 27 US 2008/0050721 A1 S N S. () O N O S US S 9SS S. OldS. no SSS CDit : 8S N S S2 asis cE wu9 ()S : O U. S. SS. w E. SSSo S. : SS O SS V SSR LO S. CN v O S. UUUOG7 e OO S O Patent Application Publication Feb. 28, 2008 Sheet 14 of 27 US 2008/0050721 A1 O) () N O) N S. () N. O S2 O 9 al OS as a S. oo: S. CD O N. S.O) OB YO s N E G O N S : CD SS tiQ 5. cSS 2 S s a u R's S OS O SS ve 9 S. UUUOG7 le OO S O Patent Application Publication Feb. 28, 2008 Sheet 15 of 27 US 2008/0050721 A1 eyesÁIayMoo?edeHgogoayB:Ogaun61-J Joueysajou?oZqe?S„Ouol?.09}êpuO aouenbesyN?sxejdnpa?efin[uod UUOG7 le OO Patent Application Publication US 2008/0050721 A1 euuseidMeyuouyo?oayB:\/9aun61-I Joueysajo?oZqe?spapue?salbu?s?ouol?oegepuo aouenbosyN?sage6nfuod to n r Patent Application Publication Feb. 28, 2008 Sheet 17 of 27 US 2008/0050721 A1 0000|| euseidMeyuouyoyoag?;89aun61-I eouanbasyN?s9qejspapueu?s316u?s?ouolgoºgºpuo ??esqueSSJouo?00?9C] CN O v UU UOG7 e OO Patent Application Publication Feb. 28, 2008 Sheet 18 of 27 US 2008/0050721 A1 N: O (9 X CD D C O O O () 92 C) ? t i CY) CN vo O UUOG7 le OO Patent Application Publication Feb. 28, 2008 Sheet 20 of 27 US 2008/0050721 A1 30uenbes6upu?q?a??aduooyo?eaoua!!:8ºdnôl-I Messexejdnpu?! xe?dnppe?ee?u?ësues?uejouo?oeleq Patent Application Publication Feb. 28, 2008 Sheet 22 of 27 US 2008/0050721 A1 Figure 10 channel 1 from Sample 1 (SampeoO1) of duplex042503002wff Max. 10.0 time. Standards A 1. to S8 Se antisense 8 g 10 2 3 17 18. 19 20 time, min B lo: 1828g to 20.034 min from sampdet (Sampdeoot) of duplexo425030a2wiff D -8 Max 297.93 cps. 2800 duplex 2300 d -9 2400 - 1537.5 2200 2000 800 O -12 1600 D-10 1400 1 5 1200 974.5 D 11 1000 OO15 1258.0 800 so D -7 4oo 852.5 198O.OO, 200 O - . 800 s 100 11oo 120 1300 14 1so 1so 1800 1900 2 21 Azeau -o: 20 to 1.1 comintronsanpe 1 (sampdecoy of duplaxfiscan2001 waff max. 83003 cps. 8500 1137.50 1365.OO sood S-5 Sense 8 - 14 maerthu -oi:10.105 to 11.984 min from sample2 (sampdoo2yof duplodeo8o3001.wiff Max. 14e4 cpa. 1.4ea 143.00 1304 antiSense Patent Application Publication Feb. 28, 2008 Sheet 23 of 27 US 2008/0050721 A1 Figure 11 Chame 1 fromSample7 (Sampleocab) of dupled5133001 wift Max. 0.7T. A 0.63 0.65 Time = Day 6 O6 05 0.50 O45 antisense O40 0.35 0.30 0.25 O2O 0.15 CO OOS OOO 3. 4. 5 6 7 8 9 10 11 12 13 14 15 16 17 8 19 20 AMD CometerChannel 1 fromSample 1 (Sample,001) of duplex050803001,wif Max 0.7Time, 0.50 O45 0.35 0.25 0.15 0.10 0.5 OOO 30 3.5 40 4.5 50 55 6.O 65 70 75 80 85 go 95 100 105 110 115 120 Time, rin Patent Application Publication Feb. 28, 2008 Sheet 24 of 27 US 2008/0050721 A1 Figure 12 ... ...nstruction of -Q1: 8643 to 11.023min from Sample 1 (Sarpedo1) of duplex050903002 wif Max 1.0 cps. A Sense 578.25 1100 Day 3 1Ooo BT TucCACUUCGCUUCACGuGuB -- 900 N-4 +/- PO3 800 N-5 N-6 Mass reconstruction of -01; 9520 to 10,981 min from Sample 2 (SampleO02) of duplex051603001,wif Max 1.0 cps. B 200 7017.6 180 170 Day 6 160 N-1 +/- PO3 150 140 N-2 130 120 110 100 3000 3500 4000 4500 5000 5500 500 6500 7000 Mass, a Tu Patent Application Publication Feb. 28, 2008 Sheet 25 of 27 US 2008/0050721 A1 Figure 13 channel 1 from Sample 1 (Sample001) of duplex042503002 wif Max. 10.0 time, . Time, min Mass reconstruction of -C1: 17.787 to 19.833 min from Sample 15 (SampleO05a) of duplex05050. Max. 1836.9 cps. B 13848.0 1800 1700 1600 7017.0 1500 1400 1300 1200 1100 1000 900 800 700 600 6830. 500 400 300 200 100 P All - - - - - - - - Mass, am) Patent Application Publication Feb. 28, 2008 Sheet 26 of 27 US 2008/0050721 A1 Patent Application Publication US 2008/0050721 A1 ?I0,1m81,1 -----???.||Î? US 2008/0050721 A1 Feb. 28, 2008 DETECTION AND QUANTITATION OF pre-clinical research settings and are not suitable for use in NUCLEC ACID MOLECULES IN human subjects. Alternative methods often rely upon hybrid BOLOGICAL SAMPLES ization Sandwich assays that can detect single stranded oligonucleotides in a sample.
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