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Method to generate biocompatible dendritic polymers for analyte detection with multimodal labeling and signal amplification

A technology for polymers and analytes, used in biochemical equipment and methods, determination/inspection of microorganisms, analytical materials, etc.

Active Publication Date: 2020-02-14
UNIV OF SOUTHERN CALIFORNIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Most analytes have no inherent signal to be used as a detection label

Method used

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  • Method to generate biocompatible dendritic polymers for analyte detection with multimodal labeling and signal amplification
  • Method to generate biocompatible dendritic polymers for analyte detection with multimodal labeling and signal amplification
  • Method to generate biocompatible dendritic polymers for analyte detection with multimodal labeling and signal amplification

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Experimental program
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Embodiment 1

[0077] molecular mechanism

[0078] The inventors' method works in the following way: Complementary pairs of dendrimers can be used to generate dendrimers by self-assembly in a controlled manner via a chain reaction of branch migration and nucleic acid hybridization in the presence of a polymerization trigger objects (Figure 1 and figure 2 ). Triggers can be used directly or in combination with affinity ligands (Figure 3 and Figure 4 ). The nature of the affinity ligand determines the type of analyte to be detected. For example, each branch of the polymer can be used to attach a label of choice, such as a fluorophore, quantum dot, or metal-chelating polymer ( Figure 5 ). Each dendrimer contains about two hundred branches. Consequently, a large number of labels can eventually be attached to the target of interest, greatly multiplying its signal and enabling rapid and unambiguous detection. Following polymerization, labeling can be accomplished by directly hybridizing ...

Embodiment 2

[0080] Advantage

[0081] Another consideration: Certain fluorophores and other signaling labels are incompatible with hairpin oligonucleotides due to secondary structure effects (eg, steric hindrance). Complementary probe labels eliminate these effects and increase synthesis yield relative to direct attachment to long oligonucleotides that can adopt secondary structure. The inventors' approach has several advantages over other approaches capable of achieving similar goals:

[0082] -By generating dendrimers rather than bare directly labeled structures (for example, in HCR), the inventors' method provides greater flexibility and Modularity. This will enable the user to select the most appropriate marker selected for the specific situation. This provides flexibility to the user such that various detection methods that can be used orthogonally to the methods described herein provide complementary information.

[0083] - Compared to HCR, the inventors' method significantly re...

Embodiment 3

[0089] Dendritic Amplifier Design Considerations

[0090] The described method allows an extremely large number of sequence designs for marker combinations. For example, for a system with a foothold / loop of 12 nucleotides in length, a stem of 24 nucleotides in length, and branches of 15 nucleotides in length, there are 4 12 ×4 24 ×4 12 ×4 15 =4 63 =8.5×10 37 possible sequence. This incredibly large design space offers the possibility to generate very large or orthogonal systems. The inventors have established design criteria for producing an optimal scale-up system.

[0091] For example, an ideal sequence combination satisfies certain design criteria including: minimizing alternative conformations of preferred hairpin secondary structures, minimizing interactions between footholds and branches, and maximizing stem stability. In more detail, sequences are selected to stack bases in key positions (e.g., the leading edge of the foothold, the last position of the foothold ...

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PUM

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Abstract

Described herein is a method to create dendritic biocompatible polymers from pairs of complementary dendritic nucleic acid monomers in a controlled manner, using polymerization triggers. The dendriticmonomers are constituted of nucleic acids and an organic polymer capable of self-assembly. Each polymer contains approximately 200 dendrites that can be used to attach labels and constitute a biologically compatible signal amplification technology. Depending on the context this technology could be used to reveal the presence of a large variety of analytes such as specific nucleic acid molecules,small molecules, proteins, and peptides.

Description

[0001] Statement Regarding Federally Sponsored Research or Development [0002] This invention was made with government support under Award HD075605 from the National Institutes of Health. The government has certain rights in this invention. technical field [0003] Described herein are methods and compositions related to dendrimers for labeling and detecting analytes. Background technique [0004] Innovative protocols are needed to detect analytes of interest in complex mixtures. Most analytes have no inherent signal to be used as a detection label. Therefore, new technologies are urgently needed to label analytes with easily detectable labels such as chromogens or fluorophores. In addition, techniques capable of signal amplification are often preferred over direct labeling methods because they enhance the signal-to-noise ratio, which increases ease and accuracy of detection. Furthermore, the most appropriate label to employ may vary in a particular case and may depend,...

Claims

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Application Information

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IPC IPC(8): C08G83/00C12Q1/682G01N21/62G01N33/53
CPCC12Q1/682C12Q2525/301C12Q2525/313C12N15/11C12N2310/531C12Q1/6834C12Q1/6841G01N21/6428G01N2021/6439
Inventor 斯科特·E·弗雷泽西蒙·雷斯特雷波约瑟夫·P·邓纳姆
Owner UNIV OF SOUTHERN CALIFORNIA
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