Artificial ncRNA Library Using Double Stem-Loop Scaffold

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Solution Overview

Problem

Developing artificial ncRNA with desirable control patterns and cell phenotypes is challenging due to the complexity of designing artificial ncRNA and the instability of random nucleotide sequences in cellular environments, as well as the difficulty in constructing large libraries.

Innovation Solution

Creating an artificial ncRNA expression library by cloning random fragmented DNA of E. coli genome into a double stem-loop RNA scaffold, which enhances stability and allows for the screening of ncRNAs that can improve cellular resistance to stressors like phenol and cinnamaldehyde.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If random nucleotide sequences are used to construct artificial ncRNA library, then the diversity of ncRNA sequences is improved, but the stability of ncRNA in cellular environment deteriorates

Engineering Contradiction:

Improvediversity of ncRNA sequencesVSAvoidstability of ncRNA

Core Design Contradiction:

Adaptability or versatilityVSStability of the object's composition

Solution Approach 1:

The artificial ncRNA is divided into two functional segments: a stable scaffold region (double stem-loop structure) and a variable insert region (random fragmented DNA). This segmentation allows the scaffold to provide structural stability while the insert region provides sequence diversity for screening functional variants.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

Different regions of the artificial ncRNA are assigned different functional qualities: the scaffold region is designed with high stability characteristics (double stem-loop structure) while the insert region is designed with high variability (random fragmented DNA). This local differentiation resolves the contradiction between stability and diversity.

Inventive Principle:

Principle #3Local quality

2Measurement precision

If the number of random nucleotide sequences increases to screen desirable artificial ncRNA, then the screening accuracy is improved, but the difficulty of constructing the library deteriorates

Engineering Contradiction:

Improvescreening accuracyVSAvoiddifficulty of constructing library

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The scaffold structure is pre-designed and prepared before introducing the random fragmented DNA inserts. This preliminary preparation of the stable scaffold framework simplifies the subsequent library construction process while maintaining the ability to screen large numbers of variants.

Inventive Principle:

Principle #10Preliminary action

3Adaptability or versatility

If artificial ncRNA is designed with complex acting patterns to achieve desirable control, then the functionality is improved, but the design complexity deteriorates

Engineering Contradiction:

Improvefunctionality of ncRNAVSAvoiddesign complexity

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The double stem-loop scaffold acts as an intermediary structure that mediates between the simple random fragmented DNA inserts and the complex functional requirements. The scaffold provides the structural framework and stability, while the inserts provide sequence specificity, dividing the design complexity into manageable components.

Inventive Principle:

Principle #24Intermediary (Mediator)

Data Source

PatentUS10227586B2Genome-originated artificial ncRNA expression library and method of preparing the same

Publication Date: 2019.03.12 KOREA ADVANCED INST OF SCI & TECH

AI summary

The present invention relates to an artificial ncRNA expression library and a method for preparing the same, and particularly the present invention relates to a library securing the stability of the artificial ncRNA by cloning random fragmented DNA that whole genome DNA of E. coli is randomly fragmented to a middle of RNA scaffold having a double stem-loop and a method for preparing the same.