Full-Length Nucleic Acid Enrichment with Blocking Oligonucleotides
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Solution Overview
Problem
Current methods for enriching full-length single-stranded nucleic acids from oligonucleotide synthesis yield low results due to inefficiencies in coupling reactions, leading to high percentages of truncated oligonucleotides, which interfere with downstream processes like cloning and result in chimeric products when amplified by PCR or purified by PAGE.
Innovation Solution
A method involving a blocking oligonucleotide that hybridizes to the target sequence of full-length nucleic acids, protecting them from digestion by a single-strand specific 5' exonuclease, while non-target nucleic acids are digested, followed by removal of the blocking oligonucleotide using RNase or other means.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If phosphoramidite method is used for oligonucleotide synthesis, then oligonucleotides can be synthesized in 3' to 5' direction with fixed 3' terminal nucleotide, but coupling efficiency is not 100% resulting in accumulation of truncated oligonucleotides as by-product
Solution Approach 1:
The patent applies preliminary action by adding a blocking oligonucleotide before the exonuclease digestion step. This blocking oligonucleotide hybridizes to the full-length target sequence in advance, protecting it from digestion. The protection is established beforehand so that when exonuclease is added, only non-target single-stranded oligonucleotides are digested while full-length protected targets remain intact.
Solution Approach 2:
The blocking oligonucleotide serves as an intermediary that mediates between the full-length target oligonucleotides and the exonuclease. It binds to the target sequence and physically blocks the exonuclease from accessing and digesting the full-length products, while allowing the exonuclease to freely digest unprotected non-target single-stranded oligonucleotides.
2Quantity of substance
If PCR amplification is used to increase yield of full-length oligonucleotides, then concentration of FLP increases, but template switching occurs resulting in chimeric products
Solution Approach 1:
The patent extracts and removes the harmful truncated oligonucleotides from the mixture through exonuclease digestion, rather than attempting to amplify the full-length products. By selectively digesting single-stranded non-target oligonucleotides and enriching for double-stranded full-length products, the method increases FLP concentration without the template switching problems associated with PCR amplification.
3Manufacturing precision
If PAGE purification is used to remove truncated oligonucleotides, then purity of full-length product improves, but yield decreases due to losses during electrophoresis
Solution Approach 1:
The patent replaces the mechanical electrophoresis separation system with a biochemical enzymatic digestion system. Instead of using electric fields and gel matrices to separate full-length from truncated oligonucleotides (which causes mechanical losses), the method uses exonuclease enzymes to selectively digest non-target single-stranded oligonucleotides, achieving purification through specific biochemical recognition and catalysis that preserves full-length products.
4Manufacturing precision
If trityl-on purification is used to purify full-length oligonucleotides, then truncated oligonucleotides are removed, but harsh acidic conditions cause depurination reducing yield and purity
Solution Approach 1:
The patent changes the chemical parameters of the purification process by using physiological pH conditions for exonuclease digestion instead of harsh acidic conditions. The exonuclease operates at neutral to slightly alkaline pH, which prevents depurination and maintains oligonucleotide stability throughout the purification process, achieving both high purity and high yield.
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
Effectively enriches full-length nucleic acids, minimizing losses and avoiding issues with template switching, while maintaining correspondence between nucleic acid tags and sequences, suitable for cloning and vector incorporation.
Implementation Method 1
a blocking oligonucleotide, wherein the blocking oligonucleotide is capable of hybridising to the target sequence of the target single-stranded nucleic acids
Implementation Method 2
contacting the mixed population of nucleic acids of step b) with a single-strand specific 5' exonuclease; and incubating the mixed population of nucleic acids of step c) under suitable conditions for the 5' exonuclease to digest the non-target single-stranded nucleic acids
Implementation Method 3
removal of the blocking oligonucleotide using RNase or other means
Data Source
AI summary
The present invention relates to methods of enriching target single-stranded nucleic acids in a mixed population of single-stranded nucleic acids. The method involves protecting the target single-stranded nucleic acids and using a 5′ exonuclease to digest the non-target single-stranded nucleic acids. The invention also relates to methods of cloning target single-stranded nucleic acids into vectors, and to associated compositions and kits.