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Ligand Binding and Linkage00:49

Ligand Binding and Linkage

5.4K
Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked.  In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence...
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Ligand Binding Sites02:40

Ligand Binding Sites

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Proteins are dynamic macromolecules that carry out a wide variety of essential processes; however, the activities of most proteins depend on their interactions with other molecules or ions, known as ligands.
Protein-ligand interactions are quite specific; even though numerous potential ligands surround a cellular protein at any given time, only a particular ligand can bind to that protein. Moreover, a ligand binds only to a dedicated area on the surface of the protein, known as the...
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Conserved Binding Sites01:49

Conserved Binding Sites

5.0K
Many proteins’ biological role depends on their interactions with their ligands, small molecules that bind to specific locations on the protein known as ligand-binding sites. Ligand-binding sites are often conserved among homologous proteins as these sites are critical for protein function.
Binding sites are often located in large pockets, and if their location on a protein’s surface is unknown, it can be predicted using various approaches. The energetic method computationally...
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Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
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Riboswitches01:56

Riboswitches

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Riboswitches are non-coding mRNA domains that regulate the transcription and translation of downstream genes without the help of proteins. Riboswitches bind directly to a metabolite and can form unique stem-loop or hairpin structures in response to the amount of the metabolite present. They have two distinct regions – a metabolite-binding aptamer and an expression platform.
The aptamer has high specificity for a particular metabolite which allows riboswitches to specifically regulate...
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A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates
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サイト選択的リガンド選択のための変異プロファイリングによる共有結合型RNAターゲティング

Phillip Yesley1, Georgia Poulladofonou1, Danny Incarnato2

  • 1Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, Nijmegen, 6525 AJ, Netherlands.

Angewandte Chemie (International ed. in English)
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PubMed
まとめ
この要約は機械生成です。

研究者らは、新規RNA標的共有結合型プローブであるCovacilを開発した。このプローブはFMNリボスイッチを選択的に修飾し、複雑な生物学的サンプル内での精密なRNA分析を可能にする。

キーワード:
共有結合修飾核酸化学RNAプローブRNAターゲティングリボスイッチ

さらに関連する動画

Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins
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Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins
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Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli
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科学分野:

  • 化学生物学
  • 分子生物学
  • RNA治療

背景:

  • RNA標的共有結合型プローブは、RNAの構造と機能を研究するために不可欠である。
  • 特定のRNA標的に対する選択的なプローブの設計は、依然として大きな課題である。
  • FMNリボスイッチは、細菌における主要な調節要素である。

研究 の 目的:

  • 構造情報に基づいたボトムアップアプローチを利用して、RNA標的共有結合型プローブを設計する。
  • FMNリボスイッチを標的とする、高度に塩基選択的な共有結合型プローブを作成する。
  • プローブの選択性と複雑なRNA環境での応用を検証する。

主な方法:

  • 変異プロファイリングと構造情報に基づいた設計戦略を利用した。
  • Ribocilスキャフォールドに基づいた共有結合型プローブのライブラリを合成した。
  • 多様な細菌種由来のFMNリボスイッチアプタマーに対してプローブをスクリーニングした。
  • 競合的フォトアフィニティ標識および質量分析法を用いて、プローブの選択性と共有結合性を検証した。

主要な成果:

  • FMNリボスイッチアプタマーに対する高度に塩基選択的な共有結合型プローブであるCovacilを同定した。
  • Covacilは、10分以内に低マイクロモル濃度で共有結合修飾を達成する。
  • 標的化されていないプローブと比較して、塩基選択性が1,000倍以上向上した。
  • 全RNAサンプル中のFMNリボスイッチに対するCovacilの選択的反応性を確認した。

結論:

  • 開発された構造情報に基づいたアプローチにより、強力で選択的なRNA標的共有結合型プローブの設計が可能になる。
  • Covacilは、FMNリボスイッチの機能およびRNA生物学を研究するための貴重なツールを表す。
  • 全RNAにおけるCovacilの塩基選択的反応性は、トランスクリプトーム解析におけるその可能性を強調している。