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