Engineered RNA Ligase Mutations for Modified Substrate Ligation
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Solution Overview
Problem
Existing RNA ligases face challenges in efficiently ligating polynucleotide substrates, particularly those containing nucleotide analogs such as modified sugar residues and non-standard internucleoside linkages.
Innovation Solution
Engineered RNA ligase polypeptides with specific amino acid sequences showing high sequence identity to reference sequences, incorporating substitutions at defined positions to enhance ligation efficiency and specificity on polynucleotides with modified nucleotides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If T4 RNA ligase is used for ligation of polynucleotide substrates, then the ligation reaction can proceed with standard RNA and DNA substrates, but the ligation efficiency is insufficient for polynucleotides containing nucleotide analogs such as modified sugar residues and non-standard internucleoside linkages
Solution Approach 1:
The patent applies parameter changes by systematically mutating specific amino acid residues in the T4 RNA ligase active site (including positions 136, 141, 142, 170, 171, 174, 196, 202, 205, 207, 209, 221, 320, 327, 332, 333, 335, 336, 339) to alter the enzyme's substrate binding properties. These amino acid substitutions change the chemical and physical parameters of the active site to accommodate modified nucleotide substrates while maintaining catalytic function, thereby improving both ligation efficiency and adaptability to diverse polynucleotide substrates
Solution Approach 2:
The patent applies local quality by making targeted modifications specifically at the active site residues of T4 RNA ligase rather than globally altering the entire enzyme structure. This localized approach allows the enzyme to maintain its overall stability and folding while creating a specialized binding pocket that can accommodate modified sugar residues and non-standard internucleoside linkages, thus improving substrate compatibility without compromising general enzymatic function
2Productivity
If engineered RNA ligase with multiple amino acid substitutions is created, then ligation activity and product yield on modified polynucleotides are improved, but the complexity of enzyme production and characterization increases
Solution Approach 1:
The patent applies segmentation by dividing the enzyme engineering process into discrete, modular components: (1) identification of specific amino acid positions for mutation, (2) creation of individual single-mutant variants, (3) combination of mutations in multi-mutant variants, and (4) systematic characterization of each variant's activity. This segmented approach allows for controlled optimization of product yield while managing production complexity through a structured, stepwise process rather than attempting to solve all variables simultaneously
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
The engineered RNA ligases demonstrate improved activity and product yield on polynucleotides with modified nucleotides, offering enhanced performance in nucleic acid synthesis and diagnostic applications.
Implementation Method 1
RNA ligases are a family of enzymes that catalyze the joining of pieces of RNA or DNA that are adjacent to each other
Implementation Method 2
T4 RNA Ligase 1 ligates a 5′-phosphoryl nucleic acid donor to a 3′-hydroxyl nucleic acid acceptor by connecting them with a phosphodiester bond
Data Source
AI summary
The present disclosure provides engineered RNA ligases, recombinant polynucleotides encoding the engineered RNA ligases, and compositions of the engineered RNA ligases. The present disclosure further provides uses of the engineered RNA ligases for ligation of polynucleotide substrates.