Substrate Linker Cleavage for High-Yield Nucleic Acid Release
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Solution Overview
Problem
Existing methods for cleaving polynucleotides from a substrate suffer from poor yield, harsh conditions, and damage to newly synthesized polynucleotides, especially when multiple sequences are cleaved simultaneously, leading to complicated analysis and mixed oligo pools.
Innovation Solution
The use of enzymatic and chemical methods, including exposure to enzymes and treatment with aqueous base, along with photo-cleavable linkers, to cleave polynucleotides from a substrate, allowing independent release and minimizing damage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional methods are used to cleave polynucleotides from substrate, then cleavage can be achieved, but yield is poor and polynucleotide integrity is damaged
Solution Approach 1:
The cleavage process is divided into distinct stages: first enzymatic cleavage of the support linker, then chemical cleavage of the polynucleotide. This segmentation allows each step to be optimized independently, improving overall yield while protecting polynucleotide integrity through controlled, sequential action rather than harsh one-step cleavage.
Solution Approach 2:
A support linker is introduced as an intermediary element between the polynucleotide and the substrate. This linker can be selectively cleaved by enzymes under mild conditions, serving as a protective intermediate that allows the polynucleotide to be released without direct exposure to harsh cleavage conditions that would damage its integrity.
2Productivity
If multiple sequences are cleaved simultaneously, then processing efficiency increases, but analysis becomes complicated and mixed oligo pools are produced
Solution Approach 1:
The cleavage process is segmented into enzymatic linker removal and subsequent chemical polynucleotide cleavage. This allows multiple sequences to be processed simultaneously in parallel, maintaining high throughput while enabling independent control and analysis of each sequence, avoiding the formation of mixed oligo pools that complicate analysis.
3Ease of manufacture
If harsh conditions are used for cleavage, then cleavage can be achieved, but polynucleotide damage occurs
Solution Approach 1:
The support linker acts as a protective intermediary that can be cleaved by enzymes under mild, physiological conditions. This intermediate step allows the polynucleotide to be released from the substrate without subjecting it to harsh chemical conditions, thereby preventing polynucleotide damage while still achieving complete cleavage.
Solution Approach 2:
The mechanical/chemical harsh cleavage process is replaced with an enzymatic mechanism for linker removal. Enzymes provide a gentle, specific, and controllable cleavage mechanism that operates under mild conditions, substituting the need for harsh chemical or physical conditions and thereby protecting polynucleotide integrity.
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
This approach enhances the yield and efficiency of polynucleotide cleavage while preserving their integrity, enabling access to specific sequences for various applications and simplifying analysis.
Implementation Method 1
exposing the plurality of polynucleotides to one or more enzymes
Implementation Method 2
one or more enzymes of the one or more enzymes comprises one or more of uracil DNA glycosylase, apurinic/apyrimidinic (AP) endonuclease, alkylpurine glycosylases C and D, OGG1, NTH1, NEIL1-3, Endonuclease V, or endonuclease VII
Implementation Method 3
treating the plurality of polynucleotides in an aqueous base at a temperature of about 55 degrees Celsius to 75 degrees Celsius
Implementation Method 4
irradiating the plurality of polynucleotides... the photo-cleavable linker is cleaved by irradiating the support linker at about 312 nm, 365 nm or 405 nm
Data Source
AI summary
Disclosed herein are methods and compositions for cleavage of nucleic acids from a surface of a solid support. Further described herein are cleavage methods compatible with enzymatic and chemical nucleic acid synthesis methods.