Oligonucleotide Adapter for Single-Stranded Nucleic Acid Library Generation
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Solution Overview
Problem
Current methods for studying protein-nucleic acid interactions, particularly protein-RNA interactions, are labor-intensive, prone to contamination, and often require radioactivity, making them inefficient and time-consuming, while existing adapters are not suitable for generating libraries from single-stranded nucleic acids.
Innovation Solution
An oligonucleotide with a double-stranded DNA portion, a non-nucleic acid spacer, and a single-stranded overhang, designed to serve as an adapter for sequencing libraries, allowing for reverse transcription and amplification without the need for linearization, and equipped with features such as barcode sequences and modified nucleotides for compatibility with sequencing kits.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional cloning methods are used for single-stranded nucleic acids, then library generation is possible, but the process is labor-intensive and requires radioactivity
Solution Approach 1:
The adapter oligonucleotide is divided into distinct functional segments: a double-stranded DNA portion for stable binding, a single-stranded overhang for ligation to the nucleic acid of interest, and a loop region containing a non-nucleic acid spacer. This segmentation allows each part to perform its specific function efficiently, enabling high-throughput library generation without complex protocols
Solution Approach 2:
The adapter oligonucleotide serves as an intermediary molecule that bridges the single-stranded nucleic acid of interest and the sequencing platform. It provides necessary functional groups for ligation, reverse transcription, and amplification, eliminating the need for radioactivity and simplifying the overall protocol while maintaining high productivity
2Adaptability or versatility
If existing adapters are used, then sequencing is possible, but they are not suitable for generating libraries from single-stranded nucleic acids
Solution Approach 1:
The adapter oligonucleotide is designed with multi-functionality to work universally with single-stranded nucleic acids. It combines ligation capability, reverse transcription priming, and amplification compatibility within a single molecule, making it adaptable for various sequencing platforms while ensuring reliable library generation from single-stranded templates
3Quantity of substance
If standard cloning protocols are used, then DNA fragments can be sequenced, but contamination from other RNAs occurs and positional information is lost
Solution Approach 1:
The adapter design incorporates local quality optimization by placing specific functional elements at precise locations: the double-stranded portion at one end for stable binding, the single-stranded overhang for directional ligation, and the loop region with barcode capacity for tracking. This spatial arrangement ensures high data quality by preventing contamination and preserving positional information while maintaining high throughput
Data Source
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AI summary
The present invention relates to an oligonucleotide comprising (a) a double-stranded portion, which double-stranded portion is DNA and 9 to 30 base in length; (b) a loop connecting the 3' end of the first strand of said double-stranded portion with the 5' end of the second strand of said double-stranded portion, said loop comprising, in 5' to 3' direction: (ba) a first DNA portion which is 4 to 20 nucleotides in length; (bb) a non-nucleic acid spacer which (i) does not interfere with the formation of a stem-loop by said oligonucleotide; and (ii) causes polymerases to cease; and (be) a second DNA portion which is 4 to 20 nucleotides in length; wherein said first DNA portion and said second DNA portion are not complementary to each other; (c) a single-stranded overhang at its 5' end, said overhang being 5 to 40 nucleotides in length, wherein (ca) the bond connecting said double-stranded portion with the nucleotide of said overhang which is directly adjacent to said double-stranded portion is cleavable under alkaline conditions; and (cb) said overhang optionally comprises a barcode sequence, said barcode sequence preferably being 5 to 10 nucleotides in length; and optionally (d) within one, more or all of (a), (b) and (c), one, more or all of the following: (da) one or more modified nucleotides; (db) one or more sequences conferring compatibility with nucleic acid sequencing kits, such compatibility being preferably the presence of regions within said oligonucleotide which are complementary to primers comprised in said sequence kits; and (dc) one or more random bases.