Controlling Gene-Silencing with Azobenzene-Containing siRNAs (siRNAzos)

Q4 Chemistry

Current Protocols in Nucleic Acid Chemistry Pub Date : 2020-11-11 DOI:10.1002/cpnc.119

Matthew L. Hammill, Golam Islam, Jean-Paul Desaulniers

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引用次数: 4

Abstract

This article contains the detailed biophysical characterization, biological testing, and photo-switching protocols of azobenzene containing siRNAs (siRNAzos), which have photoswitchable properties that can be controlled with light. First, the siRNAzos are characterized by annealing the sense and anti-sense strands together and then measuring the circular dichroism (CD) profile, and the melting temperatures (T_m) of the duplexes. Second, the biological testing of the siRNAzos in cell culture is done to determine their gene silencing efficacy. Finally, their gene-silencing activities are measured after exposure to ultraviolet (UV) light in order to inactivate the siRNAzo, and then broadband visible light, which re-activates the siRNAzo. This inactivation/reactivation protocol can be done in real time, and is reversible and robust and can be performed multiple times on the same sample if desired. © 2020 Wiley Periodicals LLC.

Basic Protocol 1: Bio-physical characterization of siRNAzo duplexes

Basic Protocol 2: Evaluation of azobenzene gene-silencing using Firefly Luciferase

Basic Protocol 3: Evaluation of azobenzene gene-silencing using reverse transcriptase-polymerase chain reaction (RT-PCR)

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含偶氮苯sirna (siRNAzos)调控基因沉默

本文包含了含偶氮苯的sirna (siRNAzos)的详细生物物理特性，生物测试和光开关协议，这些sirna具有光开关特性，可以用光控制。首先，通过将正义链和反义链一起退火，然后测量其圆二色性(CD)和熔融温度(Tm)来表征siRNAzos。其次，在细胞培养中进行siRNAzos的生物学测试，以确定其基因沉默效果。最后，在暴露在紫外线(UV)光下以使siRNAzo失活，然后在宽带可见光下重新激活siRNAzo后测量它们的基因沉默活性。这种失活/再激活方案可以实时完成，并且可逆且稳健，如果需要，可以在同一样品上多次执行。©2020 Wiley期刊有限公司基本方案1:siRNAzo双链物的生物物理特性基本方案2:利用萤火虫荧光素评价偶氮苯基因沉默基本方案3:利用逆转录-聚合酶链反应(RT-PCR)评价偶氮苯基因沉默

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Current Protocols in Nucleic Acid Chemistry Chemistry-Organic Chemistry

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期刊介绍： Published in association with International Society for Nucleosides, Nucleotides & Nucleic Acids (IS3NA) , Current Protocols in Nucleic Acid Chemistry is equally valuable for biotech, pharmaceutical, and academic labs. It is the resource for designing and running successful research projects in the rapidly growing and changing field of nucleic acid, nucleotide, and nucleoside research.