Elucidation of Photoisomerization-Related Structural Changes in an Acrylamide-Bridged Binaphthalene–Diazene Macrocyclic Chiroptical Switch by Experimental Electronic Circular Dichroism Spectra Simulation: Role of Dispersion Corrections

2015 ◽  
Vol 119 (32) ◽  
pp. 8588-8598 ◽  
Author(s):  
Lukáš Kerner ◽  
Anna Kicková ◽  
Juraj Filo ◽  
Stanislav Kedžuch ◽  
Martin Putala
Keyword(s):  
Circular Dichroism ◽  
Structural Changes ◽  
Circular Dichroism Spectra ◽  
Electronic Circular Dichroism ◽  
Circular Dichroïsm

scholarly journals Electronic Circular Dichroism of the Cas9 Protein and gRNA:Cas9 Ribonucleoprotein Complex

2021 ◽  
Vol 22 (6) ◽  
pp. 2937
Author(s):  
Monika Halat ◽  
Magdalena Klimek-Chodacka ◽  
Jagoda Orleanska ◽  
Malgorzata Baranska ◽  
Rafal Baranski
Keyword(s):  
Circular Dichroism ◽  
Conformational Changes ◽  
Structural Changes ◽  
Electronic Circular Dichroism ◽  
Guide Rna ◽  
Cas9 Protein ◽  
Rnp Complex ◽  
Characteristic Features ◽  
Circular Dichroïsm

The Streptococcus pyogenes Cas9 protein (SpCas9), a component of CRISPR-based immune system in microbes, has become commonly utilized for genome editing. This nuclease forms a ribonucleoprotein (RNP) complex with guide RNA (gRNA) which induces Cas9 structural changes and triggers its cleavage activity. Here, electronic circular dichroism (ECD) spectroscopy was used to confirm the RNP formation and to determine its individual components. The ECD spectra had characteristic features differentiating Cas9 and gRNA, the former showed a negative/positive profile with maxima located at 221, 209 and 196 nm, while the latter revealed positive/negative/positive/negative pattern with bands observed at 266, 242, 222 and 209 nm, respectively. For the first time, the experimental ECD spectrum of the gRNA:Cas9 RNP complex is presented. It exhibits a bisignate positive/negative ECD couplet with maxima at 273 and 235 nm, and it differs significantly from individual spectrum of each RNP components. Additionally, the Cas9 protein and RNP complex retained biological activity after ECD measurements and they were able to bind and cleave DNA in vitro. Hence, we conclude that ECD spectroscopy can be considered as a quick and non-destructive method of monitoring conformational changes of the Cas9 protein as a result of Cas9 and gRNA interaction, and identification of the gRNA:Cas9 RNP complex.

Sign in / Sign up

Export Citation Format

Share Document