A library of programmable DNAzymes that operate in a cellular environment

Abstract

DNAzymes were used as inhibitory agents in a variety of experimental disease settings, such as cancer, viral infections and even HIV. Drugs that become active only upon the presence of preprogrammed abnormal environmental conditions may enable selective molecular therapy by targeting abnormal cells without injuring normal cells. Here we show a novel programmable DNAzyme library composed of variety of Boolean logic gates, including YES, AND, NOT, OR, NAND, ANDNOT, XOR, NOR and 3-input-AND gate, that uses both miRNAs and mRNAs as inputs. Each gate is based on the c-jun cleaving Dz13 DNAzyme and active only in the presence of specific input combinations. The library is modular, supports arbitrary inputs and outputs, cascadable, highly specific and robust. We demonstrate the library's potential diagnostic abilities on miRNA and mRNA combinations in cell lysate and its ability to operate in a cellular environment by using beacon-like c-jun mimicking substrate in living mammalian cells.

Figure 1. YES or AND gates demonstration in vitro.

(a) Illustration of Dz13 cleaving its proper fluorescently labeled RNA substrate. (b and c) Each panel is an illustration of YES or AND gate's operation upon presence of its inputs (upper panel); Capillary-electrophoresis demonstrates cleavage or non-cleavage of the fluorescent substrate (upper middle panel); Quantitative cleavage results of 3 independent experiments (bottom-middle panel) and Plate Reader results showing reaction kinetics (bottom panel). Each gate's results match its truth table. For YES: input A = miR155, un-proper input B = miR31; For AND: input A = miR21, input B = miR125b. Standard deviation errors from three independent experiments are shown (bars).

Figure 2. Logic gate design and implementation.

Each panel shows: upper panel, schematic gate operation in the presence of its entire set of inputs; middle panel, quantitative cleavage results of 3 independent experiments; bottom panel, reaction kinetics. The results of each gate match its truth table. (a) for NOT: input A = miR31; (this is a single input gate, only A is shown) (b) for OR: input A = miR21, input B = miR125b; (c), (e) and (f) for ANDNOT, NOR & XOR: input A = miR31, input B = miR125b; (d) for NAND: input A = miR31, input B = miR155; (g) for 3-input-AND gate, input A = miR31, input B = miR21 and input C = miR125b. In (c) & (f) only input A is shown since this is the only (c) or one of (f) the input combinations that has a positive (‘True’) result in which the complex is formed. Standard deviation errors from three independent experiments are shown (bars).

Figure 3. Expression profile of breast cancer in cell lysates.

(a) and (b) Definition of Boolean expressions representing positive breast cancer diagnosis. (c) capillary electrophoresis results for the complex Boolean expression. Experiments were performed using the same conditions as the in vitro experiments in previous figures, by replacing DDW with cell lysate (1.8 mg/ml by BCA assay), and with addition of miRNAs & myc RNA to the reaction. Only upon fulfillment of conditions which meet the requirements defined for ‘breast cancer’ the DNAzyme became active and its substrate cleaved. The design of each basic logic gate (AND, ANDNOT, YES, OR) is as shown in previous figures. Variance in the cleavage efficiency of positive conditions may be explained by the different reaction kinetics underlying each ‘active’ complex (as seen in Fig. 1 & 2). Standard deviation errors from three independent experiments are shown (bars).

Figure 4. DNAzyme-based AND gate operating within cancerous living cells.

Left panel, fluorescent view of the injected cells; Right panel, phase view of the cells during and after injection. For each inputs combination 15 cells were microinjected with the following mixtures: (1) AND gate components; (2) combination of miRNA inputs (A = miR21, B = mir125b), (3) a fluorescent-quenched substrate (red) and (4) green Dextran (70 KDa), which was used to normalize and mark injected cells. (a) Representative injected cells were imaged for 5 minutes after injection. Only when both inputs were present (A + B), the red substrate was cleaved and therefore visible. ‘scr’ represent injections of a scrambled DNAzyme's component sequence, which were otherwise identical to the normal injections; (b) Average of relative change in red fluorescence in 15 cells, 5 minutes after microinjection (change was calculated as: normalized fluorescence 5 minutes post injection minus normalized fluorescence 1 minute post injection). The design of the AND gate is as shown in Fig. 1. Standard deviation errors are shown (bars). (c) Kinetic results in living cells.