Use of biological RNA scaffolds with in vitro selection to generate robust small molecule binding aptamers for genetically encodable biosensors

A technology for combining structural domains and ligands, applied in the field of oligonucleotide libraries, can solve problems such as inability to integrate easily

Pending Publication Date: 2019-11-15
UNIV OF COLORADO THE REGENTS OF
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, a bottleneck in the development of these platforms is the availability of small molecule-binding RNA aptamers that function robustly in the cellular environment.
Although aptamers can be generated by in vitro selection against almost any desired target, many of these RNA-based aptamers cannot be easily incorporated into devices or function reliably in the cellular environment

Method used

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  • Use of biological RNA scaffolds with in vitro selection to generate robust small molecule binding aptamers for genetically encodable biosensors
  • Use of biological RNA scaffolds with in vitro selection to generate robust small molecule binding aptamers for genetically encodable biosensors
  • Use of biological RNA scaffolds with in vitro selection to generate robust small molecule binding aptamers for genetically encodable biosensors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0194] Example 1: Construction of selection libraries using information from biological RNA

[0195] Examination of small biological RNAs with multihelical packing (ie, tertiary folding) indicated two recurring structures that could be considered privileged scaffolds. The first is the H-type pseudoknot, which is ubiquitous in biological RNAs, including small ribozyme ribosomal frameshift elements in viral mRNAs, and natural and synthetic aptamers. However, from a design point of view, such folding is difficult to design. Another is the three-way junction (3WJ), which is supported by long-range tertiary interactions that organize a helical arrangement around the junction. This fold is more suitable for the design of aptamer-incorporated RNA devices because it positions a designable helical element, called the P1 helix, near the ligand-binding site normally housed in the junction.

[0196] In the three-way junction foldome, there is a large number of potential candidate select...

Embodiment 2

[0208] Example 2: Scaffold selection for 5HTP yields many potential aptamers

[0209] The target of choice is 5-hydroxy-L-tryptophan (5HTP; Figure 2A ), the direct biosynthetic precursor of serotonin, which is immobilized on a solid substrate via its carboxylate group. Seven rounds of selection were performed on each library, with counterselection for L-tryptophan and increasingly stringent washing procedures in later rounds. In SSIII selection, a conventional SELEX protocol was employed in which the affinity column was washed extensively in early rounds before competitive elution to remove non-bound RNA. Competitive elution was initially observed in round four and peaked at >50% of total input RNA in round six. GsI selection uses a less stringent protocol than is generally recommended, where approximately the final 10% of total RNA remaining on the column under competitive elution is collected for amplification in the first four rounds to increase wash stringency. Maintai...

Embodiment 3

[0218] Example 3: The densest clusters retain the scaffold structure and bind 5HTP with high selectivity

[0219] Structural scaffolds greatly facilitate the verification of the structural and interaction features of the resulting aptamers. Chemical probing of RNA structures using N-methylisatoic anhydride ("NMIA") (a technique known as "SHAPE") reveals the secondary and tertiary structure of the parental scaffold as well as ligand-dependent binding in the aptamer. Whether structural changes are preserved. In GR / SSIII selection, the NMIA reactivity patterns of the 5HTP-I and 5HTP-II aptamers changed locally in the presence of ligand within the three-way junction element, consistent with this being the ligand-binding site ( Figure 3A , Figure 19 ). However, 5HTP-III displayed changes outside of J2 / 3 in the constant region, consistent with the predicted structure of a previously described tryptophan aptamer and L-Trp binding site (Majerfeld and Yarus). Retention of the GR ...

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Abstract

Provided herein are libraries of scaffolds derived from riboswitches and small ribozymes and their methods of use. The scaffolds of the invention yield aptamers that are easily identified and characterized by virtue of the structural scaffold. The nature of the scaffold predisposes these RNAs for coupling to readout domains to engineer biosensors that function in vitro and in vivo. Biosensors, synthetic RNA agents and synthetic DNA agents, and their methods of use, are also provided.

Description

[0001] related application [0002] This application claims the benefit of priority to US Provisional Patent Application No. 62 / 432,879, filed December 12, 2016, the contents of which are hereby incorporated by reference in their entirety for all purposes. [0003] Statement Regarding Jointly Sponsored Research or Development [0004] This invention was made with government support under Grant No. CMMICHE1150834 awarded by the National Science Foundation. The government has certain rights in this invention. Background technique [0005] Allosteric RNA devices are increasingly recognized as important tools capable of monitoring enzyme evolution, optimizing engineered metabolic pathways, facilitating the discovery of new genes and modulators of nucleic acid-based therapeutics. However, a bottleneck in the development of these platforms is the availability of small molecule-binding RNA aptamers that function robustly in the cellular environment. Although aptamers can be genera...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12N15/115C07K14/195C07K14/28C07K14/32C40B40/06
CPCC07K14/195C07K14/28C07K14/32C12N15/1044C12N15/111C12N15/115C12N2310/16C12N2320/11C12N2330/31C12N15/1034C12N15/1058C40B40/06C12N2310/531C12N15/1093
Inventor R·T·贝蒂E·B·波特
Owner UNIV OF COLORADO THE REGENTS OF
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