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Rationale Design of Inhibitors based on Crystal Structures of ssDNA Bound to APOBEC

Technology #20170124

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(a) Ribbon schematic of A3A-ssDNA complex showing flipped-out target C and -1 T nucleotides; overall U-shaped binding conformation. (b) Molecular surface of A3A active site; surrounding loops color-coded, superposition of stick-models of ssDNA bound to 4 different molecules in crystal’s asymmetric unit. (c) Representative ssDNA molecule shown; nucleobases and key amino side chains from active site loops labeled. (d) Wall-eye stereo view of A3A active site; bound ssDNA molecule shown in sticks, hydrogen bonds indicated by yellow dashed lines.(a) Ribbon schematic of ssDNA-A3Bctd* complex showing the flipped-out target C (0) and -1 T, as well as the overall U-shaped binding conformation. (b) A superposition of the active site region of A3A (cyan) and A3Bctd* (magenta) with relevant ssDNA substrates (opaque from a representative A3A structure and yellow from the A3Bctd* structure) showing the near-identical positioning of the flipped-out target C and -1 T. (c-d) Composite omit 2Fo-Fc map contoured at 1.0 shown for region surrounding the target cytosine (panel c) or -1 thymine (panel d). (e) Deaminase activity of wild-type A3A on ssDNA substrates containing normal T or the indicated analogs at the -1 position demonstrating that the 5-methyl group is unconstrained structurally. Uracil DNA glycosylase (UDG) readily excises dU and 5FdU from ssDNA and accounts for the 11-nucleotide product in the absence of deamination. However, due to A3A activity on the target C and the 3'-end label, only the shorter 10-nucleotide product is apparent upon deamination and gel fractionation. (f) A wall-eye stereo view of the A3Bctd* active site and the bound ssDNA molecule shown in sticks. Hydrogen bonds are indicated by yellow dashed lines. Water molecules are represented by small red crosshairs.
Categories
Researchers
Reuben Harris, PhD
Professor, Department of Biochemistry, Molecular Biology, and Biophysics
External Link (harris.cbs.umn.edu)
Hideki Aihara, PhD
Associate Professor, Department of Biochemistry, Molecular Biology, and Biophysics
External Link (cbs.umn.edu)
Managed By
Kevin Nickels
Technology Licensing Officer 612-625-7289
Patent Protection

PCT Patent Application US20180170984A1
Publications
Structural basis for targeted DNA cytosine deamination and mutagenesis by APOBEC3A and APOBEC3B
Nature Structural & Molecular Biology , Volume 24, pages 131–139 (2017)
A fluorescent reporter for quantification and enrichment of DNA editing by APOBEC–Cas9 or cleavage by Cas9 in living cells
Nucleic Acids Research, Volume 46, Issue 14, 21 August 2018, Pages e84

DNA Base Editing Enzymes with High Specificity and Efficiency

A new method will help achieve specific DNA editing events with fewer off-target issues by using modified Cas9-APOBEC fusion polypeptides. Crystal structures for ssDNA bound APOBEC3A and APOBEC3B can be used for rational design of inhibitors to impede the evolvability of viruses and tumors and for development of APOBEC-mediated base editing reporter (AMBER) systems.

Better Tool for DNA Editing In Vivo

Better tools are needed for DNA editing in vivo. The original APOBEC1-Cas9 base editing complexes have wide “editing windows”, which are nearly as big as the >20 nucleotide single-stranded DNA region displaced by annealing of the guide-RNA that directs the editing complex. The high-resolution structural information for APOBEC3A and APOBEC3B (catalytic domain) enzymes in complex with relevant single-stranded DNA substrates provide atomic-level explanations for their strong specificity for 5’-TC-3’ dinucleotide sequences within longer single-stranded DNA substrates. They also provide strong structural rationale that has already enabled the local specificity of these enzymes to be changed to 5’-CC-3’. Structural information for these extremely efficient enzymes makes it possible to tune the enzyme to preferentially edit 5’-AC-3’ and 5’-GC-3’ dinucleotide targets. Thus, these enzymes expand the DNA editing toolkit to be able to selectively target DNA cytosine bases in any dinucleotide context. In addition, unlike the normal CRISPR system, base editing requires neither double-strand DNA cleavage nor a DNA donor template.

Complementary Technologies

APOBEC3A- and APOBEC3B (catalytic domain)-Cas9 base editing complexes may be targeted with an appropriate guide RNA to virtually any DNA target. They may also be used in combination with a fluorescent reporter for DNA editing (i.e., the APOBEC- and Cas9-mediated editing (ACE) reporter system).

Phase of Development

  • Proof of concept. Further refinement and optimization on going.

Benefits

  • Achieves specific DNA editing events with fewer off-target issues
  • Enables additional fine-tuning of editing complexes by structure-guided mutagenesis and/or high-throughput screens
  • Enables screens for chemical modifiers (inhibitors or activators) of ssDNA editing

Features

  • Relies upon high-resolution crystal structure information for ssDNA bound APOBEC3A and APOBEC3B
  • May be used alone or in combination with a fluorescent reporter for DNA editing (ACE)

Applications

  • Gene editing in living cells
  • Rational design of inhibitors to impede the evolvability of viruses and tumors
  • Use alone or in combination with a fluorescent reporter for DNA editing (i.e., the APOBEC- and Cas9-mediated editing (ACE) reporter system)


Interested in Licensing?
The University relies on industry partners to scale up technologies to large enough production capacity for commercial purposes. The license is available for this technology and would be for the sale, manufacture or use of products claimed by the issued patents. Please contact Kevin Nickels to share your business needs and technical interest in this technology and if you are interested in licensing the technology for further research and development.