PMC

Luciferase activity of the L1PA2 DGnA biosensor in response to decitabine or 5-azacytidine treatment in HeLa cells. (a) Measurements of luminescence in HeLa cells co-transfected with the two modules of the L1PA2 biosensor after a 4-days decitabine treatment. The values shown are the average of experiments performed in triplicate, and the bars represent the Standard Error. (b) Measurements of luminescence in HeLa cells co-transfected with the two modules of the L1PA2 biosensor following 4-days of 5-azacytidine treatment. The values shown are the average of experiments performed in triplicate, and the bars represent the Standard Error. Firefly luciferase luminescent signal was normalized by Renilla luciferase luminescent signal as the transfection control.

Luciferase activity of the DGnA biosensor in response to specific target DNA transfected into HeLa cells. (a) Immunoblot analysis of the His-tagged modules 1 and 2 of the biosensor expressed in HeLa cells. The empty vector is the expression vector pcDNA4/HisMax without biosensor inserts. HeLa cells transfected with empty vector (second lane) show several bands, probably representing cross-reactivity of the anti-His antibody with cellular proteins. Cells transfected with expression vectors harboring each of the two biosensor constructs show additional bands corresponding to the expected molecular weights for Module 1 (95.8 kd) and Module 2 (36.1 kd). (b) Quantitation of the protein expression of the two modules in transfected HeLa cells using a Western blot analyzed in a LiCOR instrument. The 50 kDa band with the known protein amount (15 ng) of the Smart His-tagged Protein Standard (SS) was used as the reference. (c) Measurement of luminescence in HeLa cells transiently transfected with the two modules of the biosensor along with increasing amounts of co-transfected target DNA. Firefly luciferase luminescent signal was normalized by Renilla luciferase luminescent signal as the transfection control. Tg2xAB, a puc19 vector carrying target DNA, comprising two copies of the target 1 and 2 sequences, separated by 2 bp.

The two-module design of the DGnA biosensor and characterization of DNA binding of the zinc finger protein in each module. (a) schematic diagram showing His- and NLS-tagged module 1 (ZF1_NLuc_NInt) and module 2 (ZF2_CInt_CLuc). Each module contains a pentadactyl zinc finger domain linked with one half of the split VMA intein and a split firefly luciferase fusion domain. Binding of both modules via their respective zinc finger domains to their specific L1PA2 target sites induces intein-mediated protein splicing. This reaction results in covalent reconstitution of a functional luciferase capable of generating luminescence in the presence of luciferin. NLS, nuclear localization signal. ZF1 and ZF2, zinc finger domain 1 and 2. NLuc and NInt, N-terminal half of the firefly lucifease and VMA intein fusion domain. CLuc and CInt, C-terminal half of the VMA intein and firefly fusion domain. (b) DNA target sequences used to evaluate artificial zinc finger binding. Underlined bases represent changes from L1PA2 consensus sequence. (c) Immunoblot analysis of Mbp-tagged zinc finger domains in bacterial lysates following IPTG induction. (d) ELISA analysis of bacterially expressed zinc finger protein binding to the wild-type and mutant DNA targets. Bacterial lysates containing expressed zinc finger domains were used without further purification (see section), and the control lysate was generated by an empty vector expressing only the Mbp protein. Tg, target; wt, wild-type; mt, mutant; DGnA, DNA-guided nano-assembly; VMA, vacuolar membrane ATPase; Mbp, maltose binding protein; IPTG, isopropyl-beta-D-1-thiogalactopyranoside.