TALE RVD Amino Acid Combinations for Nucleotide Targeting
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Solution Overview
Problem
Current TALE-nuclease designs rely on limited RVD/base couples, leading to inactive or weakly active molecules, and there is a need for improved RVDs that can target nucleotides at different positions to enhance specificity and flexibility in nucleic acid recognition.
Innovation Solution
Development of new RVDs that target nucleotides at positions 1 to 4 within the TALE recognition domain, increasing specificity and flexibility by using specific amino acid combinations, such as II, TI, YI, and VT, to recognize A, C, G, or T, allowing for more precise nucleic acid processing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If limited RVD/base couples (NI/A, HD/C, NN/G, NG/T) are used in TALE-nuclease designs, then the design process is simplified, but the activity and specificity of the molecules are reduced leading to inactive or weakly active molecules
Solution Approach 1:
The patent expands the RVD code by introducing new amino acid combinations at positions 12 and 13 of TALE repeats, creating novel RVD/base couples that target nucleotides at positions 1-4. This parameter change in the recognition code increases both the activity and specificity of TALE-nucleases while maintaining design feasibility through systematic characterization of RVD-DNA interactions
Solution Approach 2:
The patent develops a more universal RVD code that can target all four nucleotide bases (A, C, G, T) at multiple positions (1-4) within the TALE recognition domain. This multi-functional RVD system replaces the limited conventional code, enabling broader applicability and higher reliability across diverse genomic targets
2Ease of manufacture
If conventional RVDs are used for TALE recognition, then the design follows established protocols, but the specificity and flexibility of nucleic acid recognition are insufficient
Solution Approach 1:
The patent systematically varies the amino acid residues at positions 12 and 13 of TALE repeats to create new RVD combinations, thereby changing the recognition parameters. This allows targeting of nucleotides at positions 1-4 with improved specificity while maintaining modularity and ease of design through the expanded but systematic RVD code
3Ease of operation
If existing TALE-nuclease designs are used, then the implementation is straightforward, but off-target interactions occur and activity is reduced
Solution Approach 1:
By introducing new RVDs that target nucleotides at positions 1-4 with higher specificity, the patent improves manufacturing precision of genomic targeting. The expanded RVD code reduces off-target interactions while maintaining ease of operation through the modular and systematic nature of the new recognition code
Data Source
AI summary
The present invention relates to polypeptides and more particularly to Transcription Activator-Like Effector derived proteins that allow to efficiently target and/or process nucleic acids. The present invention also concerns methods to use these proteins. The present invention also relates to vectors, compositions and kits in which RVD domains and Transcription Activator-Like Effector (TALE) proteins of the present invention are used.