Gold Nanoparticle-DNA Conjugates: An Enzymatic DNA Synthesis Platform

Abstract

Leveraging the well-established properties of gold nanoparticle (AuNP)-DNA conjugates, this research explored a novel methodology for controlled enzymatic DNA synthesis using gold nanoparticle (AuNP)-DNA conjugates as a solid platform. To this end, Hybrid Nanoparticles (HNPs) were meticulously engineered through the functionalization of AuNPs with rationally designed DNA initiator molecules. These initiator molecules, strategically attached to the AuNP surface, served as a physical support and starting point for DNA extension by the Terminal Deoxynucleotidyl Transferase (TdT) enzyme. The results confirmed the synthesis of homopolymeric DNA extensions on these HNPs (58.27 nm, PDI < 0.2), demonstrating the viability of HNPs as a platform for enzymatic DNA elongation. Although the growing demand for data storage suggests a potential application, this research established the foundational feasibility of enzymatic DNA synthesis on HNPs. While high-density DNA data storage requires extensive development, the demonstrated enzymatic synthesis on AuNP-DNA conjugates warrants significant further exploration for future applications in biotechnology and nanotechnology.

1. Flowchart Representing Methodological Protocol for DNA Data Storage

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Representation of the HNPs before and after DNA synthesis using the TdT enzyme, in dNTPs presence. It is essential to notice that the DNA in both cases is single-stranded. Authoral image created using Canva graphic design platform.

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Microscopic and UV–vis absorbance characterization of HNPs. (A) TEM image of the HNPs at a single magnification. (B) UV–vis spectrophotometry data illustrating the spectral changes: initial AuNPs (gold curve), after salt-aging with DNA and 300 mM NaCl (red curve), and following washing procedures (blue curve).

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Agarose gel electrophoresis demonstrating the efficiency of formation of HNPs. (A) Blue sample tray – used to detect SYBR-stained DNA. (B) White sample tray – used to observe the AuNPs. Lane 1/6: ladder 1 kilobase; 2: AuNPs with citrate coating; 3: Free initiator DNA (SH – C6- 5TU5T – M13 forward ssDNA); 4: HNPs after conjugation protocol; 5: HNPs purified after conjugation protocol. The red arrow demonstrates excess DNA in the conjugation protocol, the blue arrow highlights the effectiveness of the washing steps in removing this residual DNA.

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Enzymatic synthesis of DNA by TdT. (A) Homopolymer synthesis using free DNA in agarose gel electrophoresis. 1: Free initiator DNA (SH – C6 – 5TU5T – M13 forward ssDNA) (red arrow); 2: Synthesis using free DNA and dNTP; 3: Synthesis using free DNA and dGTP; 4: Synthesis using free DNA and dCTP; 5: Synthesis using free DNA and dATP; 6: Synthesis using free DNA and dTTP. (B) Homopolymer synthesis using HNPs in agarose gel electrophoresis, after the DNA was released. 1: Synthesis using HNPs and dNTP; 2: Synthesis using HNPs and dGTP; 3: Synthesis using HNPs and dCTP; 4: Synthesis using HNPs and dATP; 5: Synthesis using HNPs and dTTP; 6: Free initiator DNA (SH – C6 – 5TU5T – M13 forward ssDNA) (red arrow).

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Controlled and consecutive synthesis reaction using HNPs in agarose gel electrophoresis. L: ladder 1 kilobase; 1: HNPs after one synthesis cycle - sequence coded: T; 2: HNPs after one synthesis cycle and DNA release; sequence coded: T; 3: HNPs after two synthesis cycles; sequence coded: TC; 4: HNPs after two synthesis cycles and DNA release; sequence coded: TC; 5: HNPs after three synthesis cycles; sequence coded: TCA; 6: HNPs after three synthesis cycles and DNA release; sequence coded: TCA; 7: HNPs after four synthesis cycles; sequence coded: TCAG; 8: HNPs after four synthesis cycles and DNA release; sequence coded: TCAG; 9: Free initiator DNA (SH – C6- 5TU5T – M13 forward ssDNA); 10: Free initiator DNA (SH – C6- 5TU5T – M13 forward ssDNA) after synthesis using dNTP; 11: HNPs dispersion; 12: AuNPs. The red arrows represent the HNPs pattern after each cycle, and the white arrows indicate the DNA, released from HNPs, with AuNPs agglomerated in the gel well.

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Overview of Distribution of Synthesized Oligonucleotides Lengths Across Cycles. This figure displays the distribution of oligonucleotide and homopolymer lengths obtained from sequencing data for each synthesis cycle (2, 3, and 4). The x-axis represents the sequence length in nucleotides, and the y-axis indicates the corresponding sequence number recovered from the fourth cycle. Each horizontal line represents an individual sequenced oligonucleotide, with its length depicted by its position on the x-axis. The different colors and letters correspond to distinct homopolymer blocks within the sequences, showcasing the diversity in their composition.

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Violin Plot of Homopolymer Length Distribution Across Synthesis Cycles. This figure illustrates the distribution of homopolymer lengths for each incorporated base (T, C, A, G) across synthesis cycles 2, 3, and 4. The y-axis represents each homopolymer in bases, while the x-axis shows a violin plot of the homopolymer frequency distribution of recovered reads for all sequenced cycles.

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Violin Plot of Homopolymer Length Distribution Between Cycles. This figure illustrates the distribution of homopolymer lengths for each incorporated base (T, C) across synthesis cycles 2, 3, and 4. The y-axis represents each homopolymer in bases, while the x-axis shows a violin plot of the homopolymer frequency distribution of recovered reads. Figure segments represent the distribution found on each specific cycle.