Polymerase-Blocking Linkages in DNA-Encoded Libraries
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Solution Overview
Problem
Current methods for synthesizing and deconvoluting large or complex DNA-encoded combinatorial libraries face challenges in achieving high yields of oligonucleotide tag ligation under diverse reaction conditions, particularly at high pH and elevated temperatures, and in accurately determining tag demographics.
Innovation Solution
The development of complexes with linkages that reduce the ability of polymerases to read or translocate through, allowing for the use of non-enzymatic methods and reduced mis-tagging by bypassing or reversing these linkages, which can be chemical or enzymatic in nature, to encode and screen DNA-encoded libraries.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If standard oligonucleotide linkages are used that are readable by polymerases, then accurate sequence determination can be achieved, but mis-tagging occurs and yields are reduced under diverse reaction conditions
Solution Approach 1:
The patent inverts the conventional approach by using linkages that are deliberately NOT readable by polymerases. Instead of designing readable linkages and accepting mis-tagging, the invention uses polymerase-blocking linkages (such as phosphorothioate, peptide nucleic acid, or locked nucleic acid linkages) that prevent polymerase misreading, thereby eliminating mis-tagging while maintaining sequence information through alternative detection methods.
Solution Approach 2:
The patent introduces an intermediary solution where the encoding information is protected from polymerase misreading by using specialized linkages. The sequence information is still accessible through other means (such as direct sequencing of the oligonucleotide tags before ligation or using primer extension methods that don't require polymerase to read through the linkage), thus mediating between the need for accurate tagging and reliable ligation.
2Loss of information
If polymerase-readable linkages are used, then complete sequence information can be captured, but the library synthesis robustness decreases under high pH and elevated temperature conditions
Solution Approach 1:
The patent changes the chemical parameters of the oligonucleotide linkages by using non-standard chemistries (phosphorothioate bonds, peptide nucleic acid backbones, locked nucleic acid structures) that have enhanced stability under diverse reaction conditions including high pH and elevated temperatures. These parameter changes maintain the ability to encode sequence information while improving robustness during library synthesis.
3Productivity
If conventional enzymatic ligation methods are used, then ligation can proceed efficiently, but mis-tagging occurs due to polymerase misreading of linkages
Solution Approach 1:
The patent converts the potential harm of polymerase misreading into a benefit by deliberately using linkages that block polymerase activity. The 'harm' of reduced polymerase readability is transformed into the 'benefit' of preventing mis-tagging, as the polymerase blocking property is used intentionally to ensure accurate tag assignment during ligation.
Data Source
AI summary
The present invention relates to complexes of oligonucleotide-encoded libraries and methods of tagging and using such libraries. In particular, the oligonucleotides and methods can include complexes having at least one linkage for which a polymerase has reduced ability to read or translocate through.