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Ligand screening to pre-miRNA 149 G-quadruplex investigated by molecular dynamics

Publication . Carvalho, Josué; Santos, Tiago; Carrilho, Rui; Sousa, Fani; Salgado, Gilmar; Queiroz, João; Cruz, Carla

Using a molecular dynamics approach, the study of the interaction between six different known ligands and a predicted pre-miRNA 149 RNA G-quadruplex (rG4) structure is reported. The stabilization of rG4 structures formed within the pre-miRNA stem-loop regions using small ligands is an attractive anticancer strategy. Particularly, miRNA-149 is upregulated in a variety of cancers such as prostate cancer and is therefore a potential target for drug development. The results show that ligands C8 and PhenDC3 interact with the rG4 structure via stacking interactions with the end G-quartets. Ligands [16]phenN2, [32]phen2N4 and pyridostatin on the other hand bind the loops/groove interface of the rG4 being H-bonding and electrostatic interactions the driving force of the interaction. The C8 precursor, C8-NH2, emphasizes the structural nuances of the rG4 short loops as the lack of a large terminal aromatic moiety produced a mixed stacking-groove binding mode. Overall, this study may help the design of specific ligands for pre-miRNA rG4 towards anticancer therapeutics development. Communicated by Ramaswamy H. Sarma.

2019Journal article

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Plasmid production and purification: An integrated experiment‐based biochemistry and biotechnology laboratory course

Publication . Santos, Tiago; Pereira, Patrícia; Queiroz, João; Cruz, Carla; Sousa, Fani

This laboratory experiment describes the production and purification of plasmid DNA for undergraduate biochemistry and biotechnology courses. This experiment performed in a one-week period includes the protocols for plasmid pVAX1-LacZ production in Escherichia coli DH5α cells and subsequent purification of supercoiled pVAX1-LacZ. Firstly, the students use a growth medium that favors the replication of the plasmid resulting in a higher plasmid production during exponential growth. Afterwards, alkaline lysis is done to disrupt the bacterial cells and recover pVAX1-LacZ plasmid. Lastly, they perform the purification of pVAX1-LacZ supercoiled isoform by L-histidine chromatography, followed by agarose gel electrophoresis to characterize the separation of supercoiled isoform from contaminants. The proposed experiment provides an opportunity for students to acquire these skills that are routinely used in biochemistry and biotechnology laboratories. © 2019 International Union of Biochemistry and Molecular Biology, 47(6):638-643, 2019.

2019Journal article

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