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(12) United States Patent (10) Patent No.: US 8,715,732 B2 Luo Et Al

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(12) United States Patent (10) Patent No.: US 8,715,732 B2 Luo Et Al US008715732B2 (12) United States Patent (10) Patent No.: US 8,715,732 B2 Luo et al. (45) Date of Patent: May 6, 2014 (54) NUCLEICACID HYDROGEL VIA ROLLING 2003/0054355 A1 3/2003 Warthoe CIRCLE AMPLIFICATION 2003/02034.86 A1 10/2003 Sabatini 2003,0224395 A1 12/2003 Jovanovich et al. 2003/0228601 A1 12/2003 Sabatini (75) Inventors: Dan Luo, Ithaca, NY (US); Jong Bum 2003/0228694 A1 12/2003 Sabatini Lee, Ithaca, NY (US); Hisakage 2004, OO14078 A1 1/2004 Xia et al. Funabashi, Koganei (JP) 2004.0109853 A1 6/2004 Mcdaniel 2004O161844 A1 8, 2004 Baker et al. (73) Assignee: Cornell University, Ithaca, NY (US) 2004,024.1759 A1 12/2004 Tozer et al. 2005/0O25797 A1 2/2005 Wang et al. 2005, OO32068 A1 2/2005 Prakash et al. (*) Notice: Subject to any disclaimer, the term of this 2005, 0079132 A1 4/2005 Wang et al. patent is extended or adjusted under 35 2005/0107870 A1 5/2005 Wang et al. U.S.C. 154(b) by 497 days. 2005, 0147973 A1 7, 2005 Knott 2005, 0164283 A1* 7/2005 Krotz et al. ....................... 435/6 2006, OO24813 A1 2/2006 Warthoe (21) Appl. No.: 12/6.52,707 2006, O121452 A1 6/2006 Dhallan 2006.0160105 A1 7/2006 Dhallan (22) Filed: Jan. 5, 2010 2006/0286570 A1 12/2006 Rowlen et al. 2007/0010702 A1 1/2007 Wang et al. (65) Prior Publication Data 2007/0048759 A1 3/2007 Luo et al. 2007.0068824 A1 3/2007 Payne et al. US 2010/O189794 A1 Jul. 29, 2010 2007/0117177 A1 5/2007 Luo et al. 2007. O148246 A1 6/2007 Luo et al. Related U.S. Application Data 2007. O148639 A1 6/2007 Chagovetz et al. 2007/0178478 A1 8, 2007 Dhallan et al. (60) Provisional application No. 61/204,385, filed on Jan. 2007/0275246 A1 1 1/2007 Payne et al. 5, 2009. 2008, OOO9420 A1 1/2008 Schroth et al. 2008/0076131 A1 3/2008 Chagovetz et al. 2008. O153081 A1 6/2008 Thunnissen et al. (51) Int. Cl. 2009/0093378 A1 4/2009 Bignell et al. A6 IK 9/14 (2006.01) 2009/O137405 A1 5/2009 Bowman et al. 46.3/00 (2006.01) 2009/O142752 A1 6/2009 Hall et al. (52) U.S. Cl. 2009. O163375 A1 6/2009 Bowman et al. USPC ................................ 424/484; 424/486; 264/4 (Continued) (58) Field of Classification Search None FOREIGN PATENT DOCUMENTS See application file for complete search history. WO WO 2010/017264 A2 2, 2010 (56) References Cited WO WO 2010/017264 A3 5, 2010 OTHER PUBLICATIONS U.S. PATENT DOCUMENTS Lizardi et al., Nature Genetics, 1998; 19:225-232.* 5,445,934 A 8, 1995 Fodor et al. U.S. Appl. No. 60/783,426, filed Mar. 17 2006, Luo et al. 5,610,289 A 3, 1997 Cook et al. Altschul, et al. Gapped BLAST and PSI-BLAST: a new generation of 5,797,801 A 8, 1998 Jacob protein database search programs. Nucl. Acids. Res. 1990; 25:3389 5,885,829 A 3/1999 Mooney et al. 3402. 5,965,721 A 10, 1999 Cook et al. Becker, et al. Molecular nanosprings in spider capture-silk threads. 6,025,482 A 2/2000 Cook et al. Nat Mater. Apr. 2003:2(4):278-83. 6,201,103 B1 3, 2001 Nielsen et al. Corpet. Multiple sequence alignment with hierarchical clustering. 6,419,709 B1 7/2002 Mao et al. Nucl. Acids. Res. 1988; 16:10881-90. 6,572,605 B1 6, 2003 Humes 6,585,763 B1 7/2003 Keilman et al. Karlin, et al. Methods for assessing the statistical significance of 6,846,625 B1 1/2005 Tally et al. molecular sequence features by using general scoring schemes. 6,855,504 B2 2/2005 Fogarty Procs. Natl. Acad. Sci. USA. 1990; 87:2264-2268. 6,902,881 B2 6, 2005 Falchuk Karlin, et al. Applications and statistics for multiple high-scoring 6.951,757 B2 10/2005 Sabatini segments in molecular sequences. Proc Natl AcadSci U S A. Jun. 15, 6,962.980 B2 11/2005 Mitcham et al. 1993:90(12):5873-7. 6,963,771 B2 11/2005 Scarantino et al. 6,965,798 B2 11/2005 Kim (Continued) 6,972,195 B2 12/2005 Xu 6,982,168 B1 1/2006 Topalian et al. Primary Examiner — Doug Schultz 7,006,870 B1 2/2006 Whitehurst et al. (74) Attorney, Agent, or Firm — Wilson Sonsini Goodrich & 7,008,634 B2 3, 2006 Cima et al. Rosati 7,008,667 B2 3, 2006 Chudzik et al. 7,013, 177 B1 3, 2006 Whitehurst et al. (57) ABSTRACT 7,179,638 B2 2/2007 Anderson et al. Methods and compositions are provided for producing 7,354,706 B2 4/2008 Rowlen et al. 7,442,506 B2 10/2008 Dhallan nucleic acid-based compositions. Methods include enzyme 7,473,551 B2 1/2009 Warthoe catalyzed or nucleic acid polymer conjugation. Compositions 7,531,305 B2 5/2009 Thunnissen et al. include nucleic acid-containing hydrogels which can be elon 2001.004943.6 A1 12/2001 Zhou et al. gated via rolling circle amplification. The hydrogels can 2002fOOO6664 A1 1/2002 Sabatini encapsulate bioactive agents for drug delivery. 2003/0022231 A1* 1/2003 Wangh et al. ..................... 435/6 2003/0044855 A1 3/2003 Anderson et al. 28 Claims, 8 Drawing Sheets US 8,715,732 B2 Page 2 (56) References Cited Subramini, et al. Expression of the mouse dihydrofolate reductase complementary deoxyribonucleic acid in simian virus 40 vectors. U.S. PATENT DOCUMENTS Mol. Cell. Biol. 1981; 1:854-864. Urlaub, et al. Isolation of Chinese hamster cell mutants deficient in 2009/0209436 A1 8/2009 Larman et al. 2010/0256O17 A1* 10, 2010 Larman et al. .................. 506/40 dihydrofolate reductase activity. Proc Natl Acad Sci U S A. 1980; 2012/0022244 A1 1, 2012 Yin 77:4216-4220. Gothelf. Materials science. LEGO-like DNA structures. Science. OTHER PUBLICATIONS Nov.30, 2012:338(6111): 1159-60.doi:10.1126/science. 1229960. Liedl, et al. Controlled trapping and release of quantum dots in a DNA switchable hydrogel. Small. Oct. 2007:3(10): 1688-93. Lee, et al. Molecularly self-assembled nucleic acid nanoparticles for Logan, et al. Adenovirus tripartite leader sequence enhances transla targeted in vivo siRNA delivery. Nat Nanotechnol. Jun. 3, tion of mRNAs late after infection. Proc Natl Acad Sci U S A. Jun. 2012;7(6):389-93. doi:10.1038/nnano.2012.73. 1984;81(12):3655-9. Li, et al. Self-assembled multivalent DNA nanostructures for Pearson, et al. Improved tools for biological sequence comparison. noninvasive intracellular delivery of immunostimulatory CpG Procs. Natl. Acad. Sci. USA. 1988: 85:2444-2448. oligonucleotides. ACS Nano. Nov. 22, 2011:5(11):8783-9. doi: Scahill, et al. Expression and characterization of the product of a 10.1021/nn20277.4x. Epub Oct. 17, 2011. human immune interferon cDNA gene in Chinese hamster ovary cells. Proc Natl Acad Sci U S A. Aug. 1983:80(15):4654-8. * cited by examiner U.S. Patent May 6, 2014 Sheet 1 of 8 US 8,715,732 B2 Polymerase xx :::::::: xx : x exix xxx xxxx 8x : : x. Hydrogel 8x::::::::: FIG. U.S. Patent May 6, 2014 Sheet 2 of 8 US 8,715,732 B2 FG, 2 U.S. Patent May 6, 2014 Sheet 3 of 8 US 8,715,732 B2 wasaas S.xxxx. session 8 t | O 20 43 808 23 .C. 8EC 80 3C 2. fine min} F.G. 3 U.S. Patent May 6, 2014 Sheet 4 of 8 US 8,715,732 B2 & x & FG. 4 U.S. Patent May 6, 2014 Sheet 5 of 8 US 8,715,732 B2 FIG.S. U.S. Patent May 6, 2014 Sheet 6 of 8 US 8,715,732 B2 tö00 to 1400 1200 6 O s O SC CO 5 2 25 3CO 35 Time (h) F.G. 6 U.S. Patent May 6, 2014 Sheet 7 of 8 US 8,715,732 B2 FIG. 7 U.S. Patent May 6, 2014 Sheet 8 of 8 US 8,715,732 B2 d . SO 6 O 40 O. Ca s 8. Ratio of diameter (needle f gel) FIG. 8 US 8,715,732 B2 1. 2 NUCLECACID HYDROGEL VIA ROLLING ments of the invention, the circular nucleic acid template CIRCLE AMPLIFICATION and/or amplification products are amplified with d29 DNA polymerase. The application claims the benefit of U.S. Provisional In another aspect, the invention provides a method for Patent Application No. 61/204,385 to Luo, filed on Jan. 5, forming a hydrogel, comprising entangling one or more of a 2009, which is entirely incorporated herein by reference. first single-stranded nucleic acid molecule and a second single-stranded nucleic acid molecule, wherein the first SEQUENCE LISTING single-stranded nucleic acid molecule is formed by amplify ing a circular nucleic acid template. The instant application contains a Sequence Listing which 10 In some embodiments, the first single-stranded nucleic acid molecule is formed by amplifying a circular nucleic acid has been submitted via EFS-Web and is hereby incorporated template using a first primer. The first primer can be comple by reference in its entirety, Said ASCII copy, created on Mar. mentary to a portion of the circular nucleic acid template. 23, 2010, is named 32497718.txt and is 1,311 bytes in size. In some embodiments, the second single-stranded nucleic BACKGROUND OF THE INVENTION 15 acid molecule is formed by amplifying the first single Stranded nucleic acid molecule using a second primer.
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