{"title":"设计深红色发射性强制插层诱导发光肽作为 HIV-1 TAR RNA 配体检测的指示剂:将苯并[c,d]吲哚喹啉(BIQ)氰胺染料整合到 Tat 肽中。","authors":"Akunna Francess Ujuagu, Yusuke Sato, En Ting Tabitha Lee, Seiichi Nishizawa","doi":"10.1007/s44211-024-00642-3","DOIUrl":null,"url":null,"abstract":"<div><p>We report on a deep-red emissive fluorogenic peptide probe for human immunodeficiency virus-1 (HIV-1) trans-activation responsive (TAR) RNA as an indicator for fluorescence indicator displacement (FID) assay. The probe design is based on the concept of the forced intercalation of thiazole orange (TO) dyes (FIT) on the peptide backbone, as recently proposed by our group, where the Q (glutamic acid) residue in the Tat peptide (RKKRR-Q-RRR) is replaced with TO as if it were an amino acid surrogate. Here, instead of green emissive TO, we utilized a deep-red emissive benzo[<i>c</i>,<i>d</i>]indole-quinoline (BIQ) cyanine dye developed previously by our group for imaging of nucleolar RNA in living cells. The developed 9-mer FIT peptide (RKKRR-BIQ-RRR; named BIQ-FiLuP) exhibits a significant off–on signaling ability for TAR RNA (λ<sub>em</sub> = 660 nm, <i>I</i>/<i>I</i><sub>0</sub> = 130-fold, Φ<sub>free</sub> = 0.0009, Φ<sub>bound</sub> = 0.052), and the dissociation constant <i>K</i><sub>d</sub> reaches ca. 1 nM. When used in FID assay, BIQ-FiLuP, like TO-based FiLuP, is able to distinguish between competitive and noncompetitive inhibitors, which has never been demonstrated with all previous indicators for TAR RNA. Deep-red emissive BIQ-FiLuP facilitates the evaluation of green to yellow emissive ligands without suffering from optical interference. The combination use with green emissive TO-based FiLuP (λ<sub>em</sub> = 541 nm) would cover the examination of a wide range of fluorescent test compounds.</p><h3>Graphical abstract</h3><div><figure><div><div><picture><source><img></source></picture></div></div></figure></div></div>","PeriodicalId":7802,"journal":{"name":"Analytical Sciences","volume":null,"pages":null},"PeriodicalIF":1.8000,"publicationDate":"2024-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Design of deep-red emissive forced intercalation-induced light-up peptide as an indicator for the HIV-1 TAR RNA-ligand assay: integration of benzo[c,d]indole-quinoline (BIQ) cyanine dye into Tat peptide\",\"authors\":\"Akunna Francess Ujuagu, Yusuke Sato, En Ting Tabitha Lee, Seiichi Nishizawa\",\"doi\":\"10.1007/s44211-024-00642-3\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>We report on a deep-red emissive fluorogenic peptide probe for human immunodeficiency virus-1 (HIV-1) trans-activation responsive (TAR) RNA as an indicator for fluorescence indicator displacement (FID) assay. The probe design is based on the concept of the forced intercalation of thiazole orange (TO) dyes (FIT) on the peptide backbone, as recently proposed by our group, where the Q (glutamic acid) residue in the Tat peptide (RKKRR-Q-RRR) is replaced with TO as if it were an amino acid surrogate. Here, instead of green emissive TO, we utilized a deep-red emissive benzo[<i>c</i>,<i>d</i>]indole-quinoline (BIQ) cyanine dye developed previously by our group for imaging of nucleolar RNA in living cells. The developed 9-mer FIT peptide (RKKRR-BIQ-RRR; named BIQ-FiLuP) exhibits a significant off–on signaling ability for TAR RNA (λ<sub>em</sub> = 660 nm, <i>I</i>/<i>I</i><sub>0</sub> = 130-fold, Φ<sub>free</sub> = 0.0009, Φ<sub>bound</sub> = 0.052), and the dissociation constant <i>K</i><sub>d</sub> reaches ca. 1 nM. When used in FID assay, BIQ-FiLuP, like TO-based FiLuP, is able to distinguish between competitive and noncompetitive inhibitors, which has never been demonstrated with all previous indicators for TAR RNA. Deep-red emissive BIQ-FiLuP facilitates the evaluation of green to yellow emissive ligands without suffering from optical interference. The combination use with green emissive TO-based FiLuP (λ<sub>em</sub> = 541 nm) would cover the examination of a wide range of fluorescent test compounds.</p><h3>Graphical abstract</h3><div><figure><div><div><picture><source><img></source></picture></div></div></figure></div></div>\",\"PeriodicalId\":7802,\"journal\":{\"name\":\"Analytical Sciences\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":1.8000,\"publicationDate\":\"2024-08-05\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Analytical Sciences\",\"FirstCategoryId\":\"92\",\"ListUrlMain\":\"https://link.springer.com/article/10.1007/s44211-024-00642-3\",\"RegionNum\":4,\"RegionCategory\":\"化学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"CHEMISTRY, ANALYTICAL\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Analytical Sciences","FirstCategoryId":"92","ListUrlMain":"https://link.springer.com/article/10.1007/s44211-024-00642-3","RegionNum":4,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"CHEMISTRY, ANALYTICAL","Score":null,"Total":0}
引用次数: 0
摘要
我们报告了一种用于人类免疫缺陷病毒-1(HIV-1)反式活化反应(TAR)RNA 的深红色发射性荧光肽探针,作为荧光指示剂位移(FID)检测的指示剂。探针的设计基于我们小组最近提出的在肽骨架上强制插层噻唑橙(TO)染料(FIT)的概念,其中 Tat 肽(RKKRR-Q-RRR)中的 Q(谷氨酸)残基被 TO 取代,就像氨基酸替代物一样。在这里,我们使用了一种深红色发光的苯并[c,d]吲哚喹啉(BIQ)青染料,而不是绿色发光的 TO,这种染料是我们小组以前开发的,用于活细胞核 RNA 的成像。所开发的 9-mer FIT 肽(RKKRR-BIQ-RRR;命名为 BIQ-FiLuP)对 TAR RNA 具有显著的离体信号转导能力(λem = 660 nm,I/I0 = 130-fold,Φfree = 0.0009,Φbound = 0.052),解离常数 Kd 达到约 1 nM。在 FID 检测中使用时,BIQ-FiLuP 与基于 TO 的 FiLuP 一样,能够区分竞争性和非竞争性抑制剂,这是以往所有 TAR RNA 检测指标从未证明过的。深红色发射型 BIQ-FiLuP 可在不受光学干扰的情况下评估绿色至黄色发射型配体。与基于 TO 的绿色发射型 FiLuP(λem = 541 nm)结合使用,可检测多种荧光测试化合物。
Design of deep-red emissive forced intercalation-induced light-up peptide as an indicator for the HIV-1 TAR RNA-ligand assay: integration of benzo[c,d]indole-quinoline (BIQ) cyanine dye into Tat peptide
We report on a deep-red emissive fluorogenic peptide probe for human immunodeficiency virus-1 (HIV-1) trans-activation responsive (TAR) RNA as an indicator for fluorescence indicator displacement (FID) assay. The probe design is based on the concept of the forced intercalation of thiazole orange (TO) dyes (FIT) on the peptide backbone, as recently proposed by our group, where the Q (glutamic acid) residue in the Tat peptide (RKKRR-Q-RRR) is replaced with TO as if it were an amino acid surrogate. Here, instead of green emissive TO, we utilized a deep-red emissive benzo[c,d]indole-quinoline (BIQ) cyanine dye developed previously by our group for imaging of nucleolar RNA in living cells. The developed 9-mer FIT peptide (RKKRR-BIQ-RRR; named BIQ-FiLuP) exhibits a significant off–on signaling ability for TAR RNA (λem = 660 nm, I/I0 = 130-fold, Φfree = 0.0009, Φbound = 0.052), and the dissociation constant Kd reaches ca. 1 nM. When used in FID assay, BIQ-FiLuP, like TO-based FiLuP, is able to distinguish between competitive and noncompetitive inhibitors, which has never been demonstrated with all previous indicators for TAR RNA. Deep-red emissive BIQ-FiLuP facilitates the evaluation of green to yellow emissive ligands without suffering from optical interference. The combination use with green emissive TO-based FiLuP (λem = 541 nm) would cover the examination of a wide range of fluorescent test compounds.
期刊介绍:
Analytical Sciences is an international journal published monthly by The Japan Society for Analytical Chemistry. The journal publishes papers on all aspects of the theory and practice of analytical sciences, including fundamental and applied, inorganic and organic, wet chemical and instrumental methods.
This publication is supported in part by the Grant-in-Aid for Publication of Scientific Research Result of the Japanese Ministry of Education, Culture, Sports, Science and Technology.