Four experimenters are required: two for the biological component (DNA origami) and two for the chemical component (Gold nanorods). The biological and chemical aspects will proceed concurrently. The first 30 days are dedicated to the synthesis of DNA origami and the DNA modification of Gold nanorods (AuNRs). The subsequent 30 days focus on the DNA origami–AuNRs conjugation and photothermal switch validation.

1. DNA Origami folding, purification and charaterization

The DNA origami work includes synthesis, purification, and characterization, requiring approximately two people and about 30 days. Extensive literature can guide the synthesis experiments.^[1]

1.1 In the synthesis stage, preliminary experiments are conducted to determine optimal conditions for DNA origami formation, such as Mg²⁺ concentration. Each synthesis cycle takes roughly five days, with 2-3 cycles (around 15 days in total) typically needed to produce well-structured DNA origami.

1.2 Purification is a shorter process, taking about two days. However, the methods and reagent ratios need to be optimized, often requiring 2-3 additional trials. If materials are available in sufficient quantities, 3-4 days should be enough to yield high-quality DNA origami samples.

1.3 Characterization involves both agarose gel electrophoresis and TEM imaging. Agarose gel electrophoresis is relatively straightforward and typically achieves distinct bands after 2-3 repetitions. TEM imaging, however, is more complex, requiring careful attention to sample quality, concentration, and especially negative staining techniques. Achieving optimal TEM images usually necessitates 5-6 experimental runs, taking about 10 days in total.

2. AuNRs synthesis, ligand exchanging, DNA modification.

AuNRs are primarily divided into three parts: the synthesis and characterization of AuNRs with a length of 80 nm and a diameter of 20 nm, the substitution and characterization of surface ligands on AuNRs, and the modification and characterization of DNA on the surface of AuNRs^[2].

2.1 The synthesis cycle of AuNRs, takes approximately 3 days, including the synthesis of seeds, the growth of AuNRs, and the testing of the concentration of AuNRs solutions through UV-Vis-NIR scanning^[3][4][5]. The size and distribution of the synthesized AuNRs are tested using Dynamic Light Scattering (DLS) and transmission electron microscopy (TEM). During the experimental operation, various undesirable outcomes may appear , such as AuNRs not meeting size requirements, a wide size distribution, and a high content of spherical nanoparticles^[6]. It is necessary to explore the conditions and continuously adjust the experimental techniques and proficiency. This process is relatively time-consuming and has a higher degree of uncontrollability, with an estimated period of 30 days, going through ten experimental cycles.

2.2 The experimental cycle for the ligand exchange on the surface of AuNRs takes approximately 3 days, including the replacement of citrate ligands with BSPP ligands and the characterization after the ligand exchange.^[7] During the experimental process, undesirable outcomes such as degradation of AuNRs and aggregation may occur, which require further investigation to address. The second part of the experiment is expected to take 15 days, which takes five experimental cycles.

2.3 Modification and characterization of DNA on the surface of AuNRs. This step is relatively straightforward, thus saving time.^[8] During the experimental process, issues such as aggregation of AuNRs may arise, necessitating the screening and exploration of solutions. The experimental cycle for the modification and characterization of DNA on the surface of AuNRs is approximately 2 days, with an estimated total time of 10 days, going through five experimental cycles.

3. DNA Origami-AuNRs conjugates and characterization.

The procedures for conjugating DNA origami with AuNRs are relatively brief. However, determining the optimal molar ratio between AuNRs and DNA origami requires some exploration. Protocols from similar studies on DNA origami-AuNR assemblies can serve as useful references.^[9]

3.1 DNA Origami-AuNR Conjugation is relatively quick, but finding an optimal annealing protocol tailored to different structures is necessary and typically takes about five days.

3.2 Agarose gel electrophoresis is straightforward and usually completed within 2-3 runs over the course of 3-4 days.

3.3 In TEM experiments, sample concentration and staining techniques are critical to achieving high-quality images. This part involves refining the staining techniques and sample concentrations and typically requires 4-5 trials over approximately 10 days to produce clear TEM images.

4. Fluorescence Validation of Photothermal Switching

This phase involves using near-infrared (NIR) light to heat the AuNRs, triggering conformational changes in the DNA origami that separate the Cy5 fluorescent and BHQ2 quencher molecules, leading to fluorescence. This innovative aspect of the project may require additional time for validation. A key challenge lies in determining the optimal NIR illumination duration and power to ensure that the AuNRs induce opening in the DNA origami structure while maintaining its stability. Literature on the photothermal properties of AuNRs provides useful references for the appropriate heating power.^[10]

4.1 In practice, fluorescence measurements require a high concentration of DNA origami to ensure a detectable fluorescence signal, which necessitates substantial raw materials. Verifying the structural change in DNA origami (Doara) under NIR illumination and observing fluorescence emission typically requires 3-4 repetitions, with each experiment taking 2-3 days, totaling around 10 days for complete characterization.