Solid-Phase DNA Synthesis Using High-Quality Factor Metasurfaces

Solid-phase synthesis is the de-facto route for the synthesis of long-chained polymeric biomolecules like oligonucleotides (DNA and RNA), oligopeptides, and oligosaccharides [1,2]. Photolithographic microarrays have revolutionized solid-phase synthesis technologies by combining advances in chip scale device integration, microelectromechanical systems, and microfluidic reagent handling to enable the low-cost, high-throughput and massively parallel synthesis of these oligomers. In this approach, time multiplexed projection of an incident beam of light onto millions of reaction sites on a planar synthesis substrate using digital micromirror devices (DMDs) drives light-activated site-selective synthesis of a corresponding number of unique sequences. However, these moving mechanical mirrors along with their finite size effects result in misalignment of optical beam projections with the designated synthesis spots. The resultant deletion and substitution errors limit the yield and purity of synthesis products. Efforts to mitigate these issues by spacing the synthesis spots further apart and making them larger to generate surplus redundant copies of the same strand to aid in post synthetic error correction curtail the density and diversity of synthesis.<br/>High-Q metasurfaces consisting of arrays of dielectric nanoantennas [3-5] have the potential to address the limitations of the photolithographic microarrays. Their geometry and orientation dependent wavelength and polarization response can be exploited to uniquely excite synthesis sites without resorting to moving mechanical components. The narrow spectral linewidth in high-Q metasurfaces prevents spatial and spectral crosstalk between nanoantennas, allowing for a large number of unique resonant wavelengths over a spectral range for maximum sequence diversity that are densely packed on the synthesis substrate.<br/>Here, we present the fabrication and characterization of wavelength and polarization sensitive silicon nanoantennas for long and diverse DNA synthesis. Both in simulations and experiments, we integrate absorbing gold nanostructures for resonant photothermal heating. These resonant nanoheaters have high Q-factors ranging from 300 to 500 (simulated Q-factors around 1000). As a proof-of-concept, using infrared imaging, we demonstrate the optical switching between the 20 nanoantennas by tuning the incident wavelength and polarization without resorting to error prone mechanical deflection of the incident beam. We then demonstrate the transduction of the resonant optical switching to site-selective photothermal heating by characterizing the on and off-resonant dependence of temperature on the driving optical power. This photothermal switching will be exploited to drive thermolytic deblocking reactions only at the resonant nanoheater sites. This will be confirmed through the coupling of fluorescently labeled nucleotides only to the deblocked sites catalyzed by the enzyme terminal deoxynucleotidyl transferase. This switching mechanism will be used to demonstrate multistep synthesis reactions as well as the synthesis of diverse oligo sequences across different sites. In the absence of mechanical deflection of the excitation beam, our approach can exceed synthesis efficiencies and densities of current platforms to synthesize DNA oligomers longer than 300 nucleotides at densities exceeding 10M/cm². This will open up new avenues to genetically engineer complex biological functions in synthetic biology. Furthermore, the general operating principles discussed here can be extended for the high-throughput and massively parallel synthesis of long chained polymeric biomolecules.<br/><br/>1. Kosuri et al. <i>Nat. Methods</i>, <b>11</b>, 5, 499-507, (2014).<br/>2 Hughes et al. Ellington, <i>Cold Spring Harb. Perspect. Biol</i>., <b>9</b>, 1, a023812 (2017).<br/>3. Lawrence et al., <i>Nat. Nanotech.</i> <b>15</b>, 11, 956-961 (2020).<br/>4. Hu et al. <i>Nat. Comm.</i> <b>14</b>, 4486 (2023).<br/>5. V. Dolia et al, <i>Nat. Nanotech.</i> in print (2024).