Helitron Transposase Copy Paste DNA Amplification
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current transposon systems, such as Sleeping Beauty and PiggyBac, cannot amplify the copy number of DNA cargo once inserted into the host genome, limiting their ability to duplicate and express genes effectively.
Innovation Solution
The use of a Helitron transposase, specifically the Helraiser system, which employs a copy/paste mechanism for DNA duplication, allowing for the replication and amplification of DNA sequences within cells using a Helitron transposase and terminal sequences like LTS and RTS, enabling the introduction of single or multiple copies of a DNA sequence into the genome.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If Sleeping Beauty or PiggyBac transposon systems are used, then DNA sequences can be inserted into the host genome, but the copy number of the inserted DNA cannot be amplified
Solution Approach 1:
The patent employs a copy/paste transposon mechanism where the transposon is replicated before insertion into the host genome. The RepHel protein catalyzes rolling circle replication of the transposon DNA, generating multiple copies that are then integrated into the genome. This copying mechanism directly resolves the limitation of previous systems by enabling amplification of the DNA cargo copy number while maintaining insertability.
2Productivity
If cut/paste mechanism is used for gene duplication, then genes can be inserted into the host genome, but the ability to replicate and amplify DNA sequences is lost
Solution Approach 1:
The patent replaces the mechanical cut/paste transposon mechanism with a biochemical copy/paste mechanism involving the RepHel protein. Instead of physically excising and reinserting the transposon, the system uses rolling circle replication catalyzed by RepHel to generate multiple DNA copies that are then integrated. This substitution of mechanism enables both replication and insertion functions simultaneously.
3Quantity of substance
If traditional transposon systems are used, then DNA can be delivered to cells, but the transcriptional regulatory signals cannot be efficiently disseminated across the genome
Solution Approach 1:
The patent performs preliminary replication of the transposon DNA using the RepHel protein before the actual insertion into the host genome. This preliminary copying action ensures that multiple copies are already generated and ready for integration, thereby efficiently disseminating transcriptional regulatory signals across the genome in a single transfection event rather than requiring multiple sequential insertions.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach facilitates the efficient replication and dissemination of transcriptional regulatory signals across the genome, enabling gene function diversification and targeted gene expression by allowing stepwise amplification of DNA cargo within cells.
Implementation Method 1
Helitron transposition displays a number of features unusual for DNA transposons... putative Helitron transposases do not contain an RNase-H like catalytic domain but encode a RepHel motif made up by a replication initiator (Rep) and a DNA helicase (Hel) domain... Rep is a nuclease domain belonging to the HUH superfamily of nucleases that are involved in catalytic reactions for endonucleolytic cleavage, DNA transfer and ligation... HUH nucleases cleave exclusively ssDNA, and have a key role in the initiation of rolling circle replication
Data Source
AI summary
The invention relates to a system and method for introducing DNA into cells. In particular, the invention relates to a method for introducing single or multiple copies of a DNA sequence or gene of interest into a cell comprising providing: a) a “copy and paste” transposase; and b) a construct comprising a DNA sequence or gene of interest flanked by a “copy and paste” transposon terminal sequence, such as an LTS or RTS. A novel “copy and paste” transposon of the Helitron family is described along with systems for using the corresponding transposase in methods for introducing DNA into cells, for example, to generate cell lines for use in protein production, cell and gene therapy or as reference standards.