Methods and compositions for synthetic RNA endonucleases

a technology of endonuclease and composition, which is applied in the field of sequence specific restriction enzymes, can solve the problems of low turnover rate of nucleic acid enzymes, high production cost, and inability to fully utilize the enzyme in vitro

Inactive Publication Date: 2017-05-18
THE UNIV OF NORTH CAROLINA AT CHAPEL HILL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The synthetic RNA endonucleases achieve site-specific cleavage of target RNAs with high specificity and efficiency, enabling applications such as gene silencing, mRNA degradation, and viral RNA detection, overcoming previous limitations in RNA manipulation and analysis.

Problems solved by technology

However, unlike DNA restriction enzymes, a protein enzyme that cleaves RNA in a sequence-specific manner has not been found in nature.
However, these nucleic acid enzymes generally have low turnover rate (with kcat around 1 min−1) compared to protein enzymes, possibly due to tight binding to their substrates.
In addition, the in vitro application of such nucleic acid enzymes is compromised by the high production cost and low stability of RNA, as well as the difficulty in controlling the folding of single stranded RNA or DNA.

Method used

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  • Methods and compositions for synthetic RNA endonucleases
  • Methods and compositions for synthetic RNA endonucleases
  • Methods and compositions for synthetic RNA endonucleases

Examples

Experimental program
Comparison scheme
Effect test

example 1

References for Example 1

[0157]1. O. Takeuchi and S. Akira, Immunol Rev 227 (1), 75 (2009).[0158]2. D. P. Bartel, Cell 136 (2), 215 (2009).[0159]3. S. J. Baker, J. L. Morris, and I. L. Gibbins, Brain Res Mol Brain Res 111 (1-2), 136 (2003).[0160]4. I. J. MacRae and J. A. Doudna, Curr Opin Struct Biol 17 (1), 138 (2007).[0161]5. J. J. Champoux and S. J. Schultz, FEBS J 276 (6), 1506 (2009).[0162]6. W. G. Scott, Curr Opin Struct Biol 17 (3), 280 (2007).[0163]7. H. Yoshida, Methods Enzymol 341, 28 (2001).[0164]8. Y. Tomari and P. D. Zamore, Genes Dev 19 (5), 517 (2005).[0165]9. T. W. Nilsen, Bioessays 25 (12), 1147 (2003).[0166]10. S. K. Silverman, Nucleic Acids Res 33 (19), 6151 (2005).

[0167]11. M. Wickens, D. S. Bernstein, J. Kimble et al., Trends Genet 18 (3), 150 (2002).[0168]12. S. D. Auweter, F. C. Oberstrass, and F. H. Allain, Nucleic Acids Res 34 (17), 4943 (2006).[0169]13. X. Wang, J. McLachlan, P. D. Zamore et al., Cell 110 (4), 501 (2002).[0170]14. C. G. Cheong and T. M. Hall...

example 2

[0184]In addition to being the organelle in energy production for eukaryotic cells, the mitochondrion plays a critical role in myriad cellular processes such as control of apoptosis and ROS (reactive oxygen species) signaling. Thus mitochondria dysfunction is linked to various diseases such as cancer, autism and age-associated neurodegenerative diseases. Most mitochondrial proteins are coded by nuclear DNA and imported into mitochondria after translation. Mitochondria have a distinct genome, and the human mitochondrial genome contains 13 protein-coding, 2 rRNA, and 22 tRNA genes (FIG. 21, Panel A). Although mitochondrial gene mutations are closely linked to various diseases, the functions of these genes are hard to study because there are limited research tools to manipulate mitochondrial gene expression. A new ASRE with a mitochondrial targeting signal has been generated that can be used to specifically silence mitochondrial gene expression by cleaving mitochondrial RNA, making it ...

example 3

References for Example 3

[0228]1. Lee J E, Cooper T A. Pathogenic mechanisms of myotonic dystrophy. Biochem Soc Trans. 2009; 37(Pt 6):1281-6.[0229]2. Turner C, Hilton-Jones D. The myotonic dystrophies: diagnosis and management. J Neurol Neurosurg Psychiatry. 2010; 81(4):358-67.[0230]3. Wheeler T M, Thornton C A. Myotonic dystrophy: RNA-mediated muscle disease. Curr Opin Neurol. 2007; 20(5):572-6.[0231]4. Mahadevan M, Tsilfidis C, Sabourin L, Shutler G, Amemiya C, Jansen G, et al. Myotonic dystrophy mutation: an unstable CTG repeat in the 3′ untranslated region of the gene. Science. 1992; 255(5049):1253-5.[0232]5. Brook J D, McCurrach M E, Harley H G, Buckler A J, Church D, Aburatani H, et al. Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3′ end of a transcript encoding a protein kinase family member. Cell. 1992; 68(4):799-808.[0233]6. Liguori C L, Ricker K, Moseley M L, Jacobsen J F, Kress W, Naylor S L, et al. Myotonic dystrophy type 2 cause...

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Abstract

The present invention provides sequence specific restriction enzymes for site-specific cleavage of RNA, as well as methods of their use.

Description

STATEMENT OF PRIORITY[0001]This application is a divisional application of, and claims priority to, U.S. application Ser. No. 13 / 805,240, filed Jan. 31, 2013, which is a 35 U.S.C. §371 national phase application of Application Serial No. PCT / US2011 / 040933, filed Jun. 17, 2011, which claims the benefit, under 35 U.S.C. §119(e), of U.S. Provisional Application Ser. No. 61 / 356,340, filed Jun. 18, 2010, the entire contents of each of which are incorporated herein by reference.STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING[0002]A Sequence Listing in ASCII text format, submitted under 37 C.F.R. §1.821, entitled 5470-561v2_ST25.txt, 139,617 bytes in size, generated on Jun. 29, 2016 and filed via EFS-Web, is provided in lieu of a paper copy. This Sequence Listing is hereby incorporated by reference into the specification for its disclosures.FIELD OF THE INVENTION[0003]The present invention is directed to sequence specific restriction enzymes for site-specific cleavage of RNA, a...

Claims

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Application Information

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Patent Type & AuthorityApplications(United States)
IPC IPC(8): C12N15/10A61K38/46C12N7/00C12N9/22C12N15/86
CPCC12N15/102C12N9/22C12N2750/14143C12N7/00A61K38/465C12N15/86C07K2319/85C12Q1/44G01N2333/08G01N2333/922C12N9/16Y02A50/30
InventorWANG, ZEFENGCHOUDHURY, RAJARSHI
OwnerTHE UNIV OF NORTH CAROLINA AT CHAPEL HILL