Carrier Peptide Fragment for Nucleolar Localization
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Solution Overview
Problem
Current methods for introducing foreign substances into eukaryotic cells, particularly into the cytoplasm and nucleolus, are not efficient and lack specificity, which is a challenge in medical treatments and research.
Innovation Solution
A method using a construct comprising a carrier peptide fragment with enhanced cell membrane permeability and nucleolar localization, specifically the amino acid sequences KKRTLRKKKRKKR, KKRTLRKRRRKKR, KKRTLRKRKRKKR, and KKRTLRKKRRKKR, which allows for the efficient transfer of foreign substances such as polypeptides, nucleic acids, dyes, and drugs into the cytoplasm and nucleolus of eukaryotic cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional cell penetrating peptides are used to introduce foreign substances into eukaryotic cells, then cell membrane permeability is achieved, but introduction efficiency into the nucleolus is insufficient
Solution Approach 1:
The patent merges the cell penetrating peptide sequence (KKRRKKRKKR) with the nucleolar localization signal sequence (KKRTLRND RKKR) to create a hybrid carrier peptide that simultaneously achieves both cell membrane permeability and nucleolar localization. This combination allows the single peptide to perform both functions that were previously separate, resolving the contradiction between general cell entry and specific nucleolar targeting.
Solution Approach 2:
The carrier peptide is constructed as a composite of two functional sequences: a cell penetrating peptide component and a nucleolar localization signal component. This composite structure integrates the properties of both parent sequences, enabling the peptide to penetrate cell membranes while also directing cargo to the nucleolus, thus achieving both improvements simultaneously.
2Ease of operation
If foreign substances are introduced into eukaryotic cells using conventional methods, then cell permeability is achieved, but cytotoxicity remains high
Solution Approach 1:
The patent modifies the amino acid sequence parameters of the carrier peptide by optimizing the balance between basic amino acids (for membrane permeability) and other residues (for reduced toxicity). The specific sequence KKRTLRNDRKKR represents an optimized parameter set that maintains cell permeability while reducing cytotoxicity compared to conventional peptides with higher basic amino acid content.
3Productivity
If basic amino acid residues are substituted in the NoLS amino acid sequence, then cell membrane permeability is improved, but charge of the peptide changes
Solution Approach 1:
The patent applies local quality by substituting basic amino acid residues at specific positions (8th and 9th residues) within the peptide sequence while maintaining other residues unchanged. This localized modification approach improves cell membrane permeability at specific sites without globally altering the peptide's charge characteristics, allowing selective optimization of permeability while preserving overall stability.
Data Source
Figure 1~2A
Figure 2B~2C
Figure 2D~2E
AI summary
According to the present disclosure, a technology for efficiently introducing a desired foreign substance from the outside of eukaryotic cells into at least the cytoplasm (and the nucleolus) of the cells is provided. A method disclosed here includes (1) a step of preparing a construct for introducing a foreign substance including a carrier peptide fragment composed of any of amino acid sequences: KKRTLRKKKRKKR, KKRTLRKRRRKKR, KKRTLRKRKRKKR and KKRTLRKKRRKKR, and a foreign substance that is bound to an N-terminal side and/or C-terminal side of the carrier peptide fragment, (2) a step of supplying the construct for introducing a foreign substance to a sample containing desired eukaryotic cells, and (3) a step of incubating the sample to which the construct for introducing a foreign substance is supplied and introducing the construct into eukaryotic cells in the sample.