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Enhanced sleeping beauty transposons, kits and methods of transposition

A technology of transposon and beauty, applied in the field of nucleic acid transposition kits

Active Publication Date: 2018-12-21
MAX DELBRUECK CENT FUER MOLEKULARE MEDIZIN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Enhanced sleeping beauty transposons, kits and methods of transposition
  • Enhanced sleeping beauty transposons, kits and methods of transposition
  • Enhanced sleeping beauty transposons, kits and methods of transposition

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Embodiment 1

[0151] result

[0152] The PAI subdomain of the SB transposase mediates primary substrate contacts

[0153]The DR of the IR / DR has a composite structure, recognized by a composite DNA-binding domain. The DNA-binding domain of the SB transposase consists of two helix-turn-helix (HTH) motifs, called PAI and RED, present in the PAX of transcription factors based on their similarity to the paired (PAIRED) domain. in the family (Izsvak Z, et al., 2002 J Biol Chem, 277:34581-8.; Czerny T, et al., 1993. Genes Dev., 7:2048-61.). Both subdomains are involved in sequence-specific DNA-binding: PAI binds to the 3'-part of the dichotomous transposase binding site represented by DR while RED interacts with its 5'-part (Izsvak Z, et al., 2002. J Biol Chem, 277:34581-8). In addition to DNA binding, PAI has previously been shown to have a protein-protein interaction interface (Izsvak Z, et al., 2002. J Biol Chem, 277:34581-8.). It is worth noting that the four DRs of SB are not homogeneous...

Embodiment 2

[0218] It has previously been demonstrated that both DNA-PKcs and ATM activity are required for efficient SB transposition (Izsvák et al., 2004, Mol Cell 13(2):279-90). Similar to DNA-PKc and ATM, ATR also belongs to the phosphatidylinositol 3-kinase-like kinase (PIKK) family and is involved in checkpoint signaling and repair. ATR is specifically activated by DNA damage during replication (Lupardus et al., 2002, Genes Dev 16(18):2327-32). Caffeine is an inhibitor of ATM, ATR and mTOR (also a member of PIKK), but not DNA-PKC (Sarkaria et al., 1999, Cancer Res. 59(17):4375-82). The inventors detected SB transposition using a standard transposition assay under caffeine treatment (4 mM).

[0219] Compared with the control, the transposition frequency was reduced by about 50% after caffeine treatment ( Figure 7A ). To elucidate whether efficient SB transposition is specifically required for ATR signaling, a stable TET-inducible cell line in which ATR function could be regulated...

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Abstract

The present invention relates to enhanced Sleeping Beauty-type transposons and methods of transposition. In particular, the invention relates to a polynucleotide comprising a cargo nucleic acid flanked by a left and a right inverted repeat / direct repeat (IR / DR), wherein IR / DRs, having specific sequences, are recognized by a Sleeping Beauty transposase protein and the polynucleotide is capable of integrating into the DNA of a cell. The invention also relates to a kit for transposing a nucleic acid comprising said polynucleotide as well as to further components such as co-factors of transposition capable of depleting a component of the FACT (facilitates chromatin transcription) complex, namely, SSRP1 and / or SUPT16H / SPT16,or an inhibitor of cathepsin selected from the group comprising H,S,V,and L; or a cofactor capable of depleting or inhibiting HSP90; or a factor temporally arresting cells cell cycle in cell cycle phase G0 / G1, G1 / S, or G2 / M; or a factor inhibiting the ubiquitination ofPCNA,or cells wherein these components have been knocked down or inhibited,or the cell cyle arrested in any of said stages. Alternatively or additionally, the kit may comprise as a co-factor of transposition an agent capable of increasing concentration and / or signaling of ATR or a cell wherein concentrationand / or signaling of ATR are increased. The invention further provides methods using said transposon polynucleotide as well as host cells and pharmaceutical compositions. It also relates to use of said co-factors of transposition or specific cells for enhancing transposition efficiencies, e.g., for preparing genetically modified nucleic acids or cells.

Description

technical field [0001] The present invention relates to enhanced Sleeping Beauty-type transposons and methods of transposition. In particular, the present invention relates to polynucleotides comprising cargo nucleic acid flanked by left and right inverted repeats / direct repeats (IR / DR), wherein the IR / DR having a specific sequence is The Sleeping Beauty transposase protein recognizes and this polynucleotide is able to integrate into the DNA of the cell. The present invention also relates to a kit for nucleic acid transposition comprising said polynucleotide and further components, such as transposition cofactors capable of eliminating components of the FACT (chromatin-activated transcription) complex, namely SSRP1 and / or or SUPT16H / SPT16, or an inhibitor of cathepsin selected from the group comprising H, S, V and L; or a cofactor capable of eliminating or inhibiting HSP90; or in cell cycle phases G0 / G1, G1 / S or factors that temporarily arrest the cell cycle in G2 / M; or fact...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N15/113
CPCC12N2800/90C12N15/90A61P31/04A61P31/12A61P35/00
Inventor 苏兹萨纳·伊日瓦克佐尔坦·伊维克斯克里斯托弗·考夫曼苏内尔·纳拉亚纳瓦里
Owner MAX DELBRUECK CENT FUER MOLEKULARE MEDIZIN
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