Rapid Enzymatic Assay for Antiretroviral Drug Monitoring Using CRISPR-Cas12a-Enabled Readout.

IF 3.9 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

ACS Synthetic Biology Pub Date : 2025-02-21 Epub Date: 2025-02-11 DOI:10.1021/acssynbio.4c00674

Maya A Singh, Megan M Chang, Qin Wang, Catherine Rodgers, Barry R Lutz, Ayokunle O Olanrewaju

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Abstract

Maintaining the efficacy of human immunodeficiency virus (HIV) medications is challenging among children because of dosing difficulties, the limited number of approved drugs, and low rates of medication adherence. Drug level feedback (DLF) can support dose optimization and timely interventions to prevent treatment failure, but current tests are heavily instrumented and centralized. We developed the REverse transcriptase ACTivity crispR (REACTR) for rapid measurement of HIV drugs based on the extent of DNA synthesis by HIV reverse transcriptase. CRISPR-Cas enzymes bind to the synthesized DNA, triggering collateral cleavage of quenched reporters and generating fluorescence. We measured azidothymidine triphosphate (AZT-TP), a key drug in pediatric HIV treatment, and investigated the impact of assay time and DNA template length on REACTR's sensitivity. REACTR selectively measured clinically relevant AZT-TP concentrations in the presence of genomic DNA and peripheral blood mononuclear cell lysate. REACTR has the potential to enable rapid point-of-care HIV DLF to improve pediatric HIV care.

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使用crispr - cas12a激活读出的抗逆转录病毒药物监测的快速酶分析。

由于给药困难、批准的药物数量有限以及药物依从率低，在儿童中维持人类免疫缺陷病毒（HIV）药物的疗效是一项挑战。药物水平反馈（DLF）可以支持剂量优化和及时干预，以防止治疗失败，但目前的测试是大量仪器化和集中的。我们开发了逆转录酶活性crispR (REACTR)，用于基于HIV逆转录酶合成DNA的程度快速测量HIV药物。CRISPR-Cas酶与合成的DNA结合，触发淬灭报告基因的侧切并产生荧光。我们检测了儿童HIV治疗的关键药物AZT-TP (azidothymidine triphosphate)，并研究了检测时间和DNA模板长度对REACTR灵敏度的影响。在基因组DNA和外周血单核细胞裂解液存在的情况下，REACTR选择性地测量临床相关的AZT-TP浓度。REACTR有潜力实现快速的即时艾滋病毒DLF，以改善儿科艾滋病毒护理。

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来源期刊

ACS Synthetic Biology 生物-

CiteScore

8.00

自引率

10.60%

发文量

380

审稿时长

6-12 weeks

期刊介绍： The journal is particularly interested in studies on the design and synthesis of new genetic circuits and gene products; computational methods in the design of systems; and integrative applied approaches to understanding disease and metabolism. Topics may include, but are not limited to: Design and optimization of genetic systems Genetic circuit design and their principles for their organization into programs Computational methods to aid the design of genetic systems Experimental methods to quantify genetic parts, circuits, and metabolic fluxes Genetic parts libraries: their creation, analysis, and ontological representation Protein engineering including computational design Metabolic engineering and cellular manufacturing, including biomass conversion Natural product access, engineering, and production Creative and innovative applications of cellular programming Medical applications, tissue engineering, and the programming of therapeutic cells Minimal cell design and construction Genomics and genome replacement strategies Viral engineering Automated and robotic assembly platforms for synthetic biology DNA synthesis methodologies Metagenomics and synthetic metagenomic analysis Bioinformatics applied to gene discovery, chemoinformatics, and pathway construction Gene optimization Methods for genome-scale measurements of transcription and metabolomics Systems biology and methods to integrate multiple data sources in vitro and cell-free synthetic biology and molecular programming Nucleic acid engineering.