DNA "nano-claw": logic-based autonomous cancer targeting and therapy

Abstract

Cell types, both healthy and diseased, can be classified by inventories of their cell-surface markers. Programmable analysis of multiple markers would enable clinicians to develop a comprehensive disease profile, leading to more accurate diagnosis and intervention. As a first step to accomplish this, we have designed a DNA-based device, called "Nano-Claw". Combining the special structure-switching properties of DNA aptamers with toehold-mediated strand displacement reactions, this claw is capable of performing autonomous logic-based analysis of multiple cancer cell-surface markers and, in response, producing a diagnostic signal and/or targeted photodynamic therapy. We anticipate that this design can be widely applied in facilitating basic biomedical research, accurate disease diagnosis, and effective therapy.

Figure 1

The symbols and construction schemes are shown for (a) 2-input trivalent “Y”-shaped Nano-Claw and (b) 3-input tetravalent “X”-shaped Nano-Claw. (c) Flow cytometry experiment to determine the best cDNA sequences with a high Cy5.5 fluorescence signal (from biotin-labeled TC01, Sgc4f or Sgc8c aptamer) and low FITC fluorescence signal (labeled on the candidate strands).

Figure 2

(a) Experimental scheme of aptamer-switch AND gate. (b) Adjusting the gating properties by changing the DNA sequence design, CEM and HeLa cells can be either dually targeted or distinguished. (c) Cell viability test for the AND and INH gates after visible irradiation for 3h and subsequent growth for 48h (*: p-value < 0.05; **: p-value <0.001; by comparison with each irradiated cell type only, n=3). (d,e) Flow cytometric analysis and confocal microscope images (TAMRA dye) of the fluorescence signal with/without the gate probes for the AND and INH gate. The fluorescence values and their error bars (mean ± s.d.) were calculated from three experiments.

Figure 3

(a) Fluorescence measurements in buffer solution and (b) flow cytometric signals prove the gating and displacement properties for the Nano-Claw, where the red-colored S’ or T’ indicates the dye-labeled strand. (c) Cell viability test for the “Y”-shaped claw, where “Q” stands for BHQ-3 quencher that prevented the Ce6-induced PDT reaction. (d,e) The signal reporting of the Nano-Claw on the CEM and Ramos cell membrane, “cX(d)” represents the fully-complementary control X/cX duplex that was labeled on one capture toe and could not release the cX strand.

Figure 4

The effect of the “poly-T” linker length on the functions of the (a) “Y”- or “X”-shaped claw with two capture toes and (b) “X”-shaped claw with three capture toes. The fluorescence values were calculated based on the distributions in the flow cytometer from three experiments.