Polydactyl zinc finger (PZF) proteins bind double-stranded DNA based on sequence-specific molecular recognition of DNA bases by helical peptide domains, which penetrate the major groove of B-DNA (Klug, 2010
). Each PZF recognizes 3
bp of DNA in a quasi-modular manner, and thereby the rational design of artificial PZFs is possible (Blancafort et al.
; Bhakta and Segal, 2010
). Just as the PCR achieves single-locus specificity based on the use of two different primers, it is possible to target a genomic locus with high specificity employing a set of two contiguous five-finger PZF modules, which together recognize 15+15=30
bp. The use of separate modules of PZF proteins to recognize specific DNA sequences, resulting in the assembly of a green fluorescent protein reporter has been described (Stains et al.
; Furman et al.
). We modified this system to be under the control of a conditional protein splicing system, in which fragments of an artificially split Saccharomyces cerevisiae
vacuolar membrane ATPase (VMA
) intein complement through rapamycin-induced heterodimerization of the FK506 binding protein (FKBP) and FKB12-Rapamycin binding domain of mTor (FBR) Proteins (Mootz and Muir, 2002
). This intein-based biosensor system was able to report the presence of the drug rapamycin in a living animal, in real time (Schwartz et al.
) meeting one of our key design requirements.
In our biosensor design, specific DGnA of a luminescent reporter is driven by two independent protein fusions each comprising a PZF module with five PZF domains, a half-intein domain, and a half-luciferase-enzyme domain. illustrates the two zinc finger biosensor structures ZF1 and ZF2, and the resulting DGnA process, in which each of the modules specifically binds at adjacent sites in the genome, followed by intein-catalyzed splicing and the generation of covalently joined and active luciferase. We targeted specific DNA sequences representing the consensus sequence (see Supplementary Fig. S1
) of the 5′-UTR of Line-1 elements belonging to the L1PA2 subfamily, a group of sequences that numbers ~4600 interspersed copies, originating in Hominoidea
, and largely absent in older species of monkeys such as Rhesus macaque
. To evaluate DNA-recognition specificity, we expressed the artificial zinc finger proteins in Escherichia coli
(), and confirmed by means of ELISA assays that each of the two PZF modules specifically binds to its cognate sequence, and not to other DNA sequences (). Note that the two strongest binding signals in correspond to the perfect L1PA2 genomic target sequences Tg1wt and Tg2wt.
FIG. 1. 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 (more ...)
We then showed that each of the two fusion proteins could be expressed in HeLa cells (), and optimized protein expression levels by adjusting the amount of transfected DNA until an expression level ranging between 56,000 and 134,000 molecules per cell () was attained for each of the protein modules. The feasibility of the DGnA process was evaluated by co-transfecting HeLa cells with an artificial DNA plasmid that harbors the entire target region corresponding to the L1PA2 consensus sequence. As shown in , transfection was performed with different amounts of target DNA or with an irrelevant DNA target, and biosensor performance was assessed as a ratio of firefly luciferase (biosensor) to Renilla
luciferase (transfection control) in a dual luciferase assay. The results, as indicated by increasing values for the luminescence ratio of firefly luciferase to Renilla
luciferase, are consistent with the two genetically encoded biosensor modules being able to bind the artificial L1PA2 sequence (denominated Tg2xAB) in the transfected targets, and undergoing conditional protein splicing to generate, via the DGnA process, molecules of covalently joined and active luciferase. We also observed a significant amount of luminescence signal for the empty (target-free) vector DNA input measurement, most likely arising from background (nonconditional) intein splicing as reported by others (Schwart et al.
, 2006) that can occur in the absence of DNA binding. Since the largest input used in this experiment (1200
ng of transfected plasmid) has been determined to correspond to ~3200 molecules of artificial target internalized in cell nuclei (see Materials and Methods
section), and this number of artificial targets is smaller than the number of L1PA2 elements in a diploid genome, it seems unlikely that background signal could be generated by a small subcomponent of undermethylated L1PA2 genomic DNA. Despite the nonconditional intein splicing background issue, the experiment shown in demonstrates a strong luminescence response in the presence of artificial DNA targets.
To demonstrate the utility of the genetically encoded biosensors in a relevant biological context we performed several experiments where cells are exposed to drugs capable of inducing DNA demethylation. The responses of mammalian cells to the demethylating drugs decitabine and 5-azacitidine have been extensively documented in the literature. To illustrate the effects of decitabine on the DNA methylation levels of L1PA2 repetitive elements in HeLa cells, in which Line-1 elements have been reported to be predominantly methylated (Teneng et al.
), we treated a HeLa cell culture with decitabine, at a concentration of 2.5
μM, for a period of 4 or 5 days. The drug induced a loss of methylation corresponding to values lower than 70% of the control HeLa cell culture methylation levels in 8 out of 21 CpG dinucleotides. The lowest observed levels of relative DNA methylation compared to controls, after decitabine treatment, were 51%, 54%, and 55% for three of the 21 CpG dinucleotides assayed (). The drug concentrations used in these and subsequent experiments were chosen to be roughly comparable to the maximum concentrations (Cmax) achieved in human plasma at clinically used dosages and schedules of administration. Human plasma Cmax values are 3 to 11
μM 5-azacytidine and 0.3 to 1.6
μM decitabine (Marcucci et al.
; Blum et al.
; Cashen et al.
). Similar drug concentrations to those used in our study have been utilized in recent cell-line based studies of these drugs (Hollenbach et al.
Table 1. Measurements of Line-1 5-UTR DNA Methylation in HeLa Cells Treated with 2.5μM Decytabine for 4 or 5 days
We performed an experiment, using triplicate cell cultures, where the biosensors were transfected into HeLa cells 3 days after the start of drug treatment. The results, shown in , demonstrate that incremental doses of decitabine lead to significant increases in luminescent output of the DGnA biosensors, demonstrating the ability of the system to report changes in accessibility of the genomic DNA harboring L1PA2 elements, as they become demethylated. We performed a similar experiment using the demethylating drug 5-azacytidine, and the results are shown in . In this second experiment, also performed in triplicate, the DGnA biosensors report the increased DNA accessibility in L1PA2 genomic DNA, resulting from 5-azacytidine treatment, showing a drug concentration-dependent increase in relative luminescent output.
FIG. 3. 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. (more ...)