Oligonucleotide Conformational Modulation for RNase H Cleavage
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Solution Overview
Problem
Current methods for optimizing the cleavage of RNA species by RNase H are limited in specificity and efficiency, particularly in modulating the helical conformation of RNA/DNA heteroduplexes, which affects the enzyme's positional preference and activity.
Innovation Solution
The development of oligonucleotides with regions of differing conformation and a transition moiety that modulates the helical conformation from the 3' hydroxy to the 5' hydroxyl, incorporating modified nucleotides and internucleotide linkages to optimize RNase H activity, including phosphorothioates and 2'-O-alkoxyalkyl ribonucleotides, to enhance cleavage specificity and rate.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If oligonucleotides with uniform conformation are used, then the structure is simple, but the RNase H cleavage specificity and efficiency are limited
Solution Approach 1:
The oligonucleotide is divided into multiple regions with different conformations (e.g., DNA-like regions, RNA-like regions, and transition regions). Each region serves a specific function: the DNA-like region provides stability, the RNA-like region facilitates RNase H binding, and the transition region enables conformational modulation. This segmentation allows the oligonucleotide to achieve both structural complexity for enhanced cleavage efficiency and functional organization.
Solution Approach 2:
Different regions of the oligonucleotide are赋予 different local conformational properties. The 3' region may adopt a DNA-like conformation for stability, while the 5' region adopts an RNA-like conformation for RNase H interaction. This local differentiation of conformational quality enables the oligonucleotide to simultaneously satisfy conflicting requirements for stability and enzyme recognition, thereby improving cleavage efficiency without requiring uniform complexity throughout the entire structure.
2Manufacturing precision
If the helical conformation of RNA/DNA heteroduplex is not modulated, then the oligonucleotide structure is simple, but the enzyme's positional preference and activity are suboptimal
Solution Approach 1:
A transition region comprising modified nucleotides (such as 2'-O-alkoxyalkyl ribonucleotides or phosphorothioates) serves as an intermediary between DNA-like and RNA-like regions. This transition region modulates the helical conformation of the heteroduplex, enabling precise positioning of the RNase H enzyme at the desired cleavage site. The intermediary elements facilitate controlled conformational changes without requiring complex overall oligonucleotide structures.
Solution Approach 2:
The oligonucleotide incorporates modified nucleotides that change the helical parameters (such as twist, rise, and tilt) of the RNA/DNA heteroduplex. By adjusting these physical parameters through conformational modulation elements, the invention achieves precise control over the enzyme's positional preference and cleavage specificity, transforming the heteroduplex geometry to optimize RNase H activity.
3Measurement precision
If modified nucleotides and internucleotide linkages are incorporated, then the cleavage specificity and rate are enhanced, but the synthesis complexity increases
Solution Approach 1:
Modified nucleotides and internucleotide linkages (such as phosphorothioates or 2'-O-alkoxyalkyl ribonucleotides) are incorporated only in specific transition regions where they are most needed for conformational modulation, rather than throughout the entire oligonucleotide. This localized modification approach maintains high cleavage specificity while reducing synthesis complexity compared to fully modified oligonucleotides.
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 optimizes the cleavage rate and position of target RNA by RNase H, improving the enzyme's efficiency and specificity, and can be used to modulate gene expression by inhibiting or upregulating targeted genes.
Implementation Method 1
a transition moiety which modulates the transmission of the conformation of said second region into said first region
Implementation Method 2
RNase H hydrolyzes RNA in RNA-DNA hybrids
Implementation Method 3
RNase H activity appears to be ubiquitous in eukaryotes and bacteria
Data Source
AI summary
The present invention provides compositions and methods for the optimization of cleavage of RNA species by RNase H. In some embodiments, the invention provides oligonucleotides that possess two or more regions of differing conformation, and at least one transitional nucleobase positioned between the regions that is capable of modulating transfer of the helical conformation characteristic of the region bound to the 3′hydroxy thereof, to the region bound to the 5′ hydroxyl thereof.