Argonaute Polypeptide Single-Stranded Nucleic Acid Targeting
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Solution Overview
Problem
Current methods for targeting Ago nuclease activity to single-stranded nucleic acids are limited due to the indistinguishability of guide RNAs from host cell RNAs, making precise and controlled targeting challenging.
Innovation Solution
The use of a subject argonaute (Ago) polypeptide and a guide RNA with a 5'-OH, which can be introduced into cells or used in vitro, to specifically bind and cleave single-stranded target nucleic acids, such as ssRNA or ssDNA, by employing variants of the Marinitoga piezophila Ago protein with reduced nuclease activity or fusion partners for modified activities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If guide RNA with 5'-phosphate is used for Ago protein targeting, then the guide RNA can be naturally processed and loaded into Ago proteins, but the guide RNA becomes indistinguishable from host cell RNAs, reducing targeting precision
Solution Approach 1:
The patent applies local quality by modifying only the 5' end of the guide RNA with a non-natural nucleotide or chemical group, while keeping the rest of the guide RNA sequence and structure natural. This localized modification enables distinction from host RNAs without compromising the overall guide RNA function and Ago protein loading capability.
Solution Approach 2:
The patent uses non-natural nucleotides or chemical modifications as intermediaries between the guide RNA and Ago protein system. These intermediaries serve as distinguishable markers that allow the Ago protein to recognize and process the guide RNA while enabling differentiation from host cell RNAs through the unique chemical signature.
2Quantity of substance
If heterologous Ago proteins are expressed in host cells, then the Ago proteins can be produced in sufficient quantities, but the guide RNAs become indistinguishable from the thousands of RNAs present in the host cell
Solution Approach 1:
The patent employs chemical modifications analogous to color changes, where non-natural nucleotides or chemical groups serve as distinctive markers on the guide RNA. This chemical 'coloring' allows the guide RNA to be visually or chemically distinguished from the background of host cell RNAs, enabling precise targeting despite the presence of numerous endogenous RNAs.
3Productivity
If Ago protein nuclease activity is enhanced for efficient cleavage, then the cleavage efficiency increases, but the specificity and control over target selection decreases
Solution Approach 1:
The patent applies dynamics by using inducible or controllable systems where the Ago protein's nuclease activity can be activated or deactivated on demand. This dynamic control allows the system to maintain high cleavage efficiency when needed while providing temporal and spatial control to ensure specificity in target selection, preventing off-target effects.
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 enables precise and controlled targeting and modification of single-stranded nucleic acids, allowing for cleavage or other enzymatic activities like methylation, thereby overcoming the limitations of existing technologies.
Implementation Method 1
Ago proteins use a guide nucleic acid (e.g., a guide RNA) to identify a target nucleic acid
Implementation Method 2
subject argonaute (Ago) polypeptide and a guide RNA... to specifically bind and cleave single-stranded target nucleic acids
Data Source
Figure 1A
Figure 1B
Figure 1B
AI summary
The present disclosure provides compositions, kits, genetically modified cells, non-human transgenic organisms, and methods for binding and/or cleaving a single stranded target nucleic acid. A method of cleaving includes contacting a single stranded target nucleic acid with (e.g., introducing into a cell) a subject argonaute (Ago) polypeptide and a guide RNA (e.g., having a 5' -OH). In some embodiments, a subject Ago polypeptide includes an amino acid sequence having 70% or more sequence identity with amino acids 282-430 and/or 431-639 of the Marinitoga piezophila argonaute (MpAgo) protein set forth in SEQ ID NO: 1. The present disclosure provides variant Ago polypeptides; and methods of use of same.