Thermal stability of DNA functionalized gold nanoparticles

Abstract

Therapeutic uses of DNA functionalized gold nanoparticles (DNA-AuNPs) have shown great potential and exciting opportunities for disease diagnostics and treatment. Maintaining stable conjugation between DNA oligonucleotides and gold nanoparticles under thermally stressed conditions is one of the critical aspects for any of the practical applications. We systematically studied the thermal stability of DNA-AuNPs as affected by organosulfur anchor groups and packing densities. Using a fluorescence assay to determine the kinetics of releasing DNA molecules from DNA-AuNPs, we observed an opposite trend between the temperature-induced and chemical-induced release of DNA from DNA-AuNPs when comparing the DNA-AuNPs that were constructed with different anchor groups. Specifically, the bidentate Au-S bond formed with cyclic disulfide was thermally less stable than those formed with thiol or acyclic disulfide. However, the same bidentate Au-S bond was chemically more stable under the treatment of competing thiols (mercaptohexanol or dithiothreitol). DNA packing density on AuNPs influenced the thermal stability of DNA-AuNPs at 37 °C, but this effect was minimum as temperature increased to 85 °C. With the improved understanding from these results, we were able to design a strategy to enhance the stability of DNA-AuNPs by conjugating double-stranded DNA to AuNPs through multiple thiol anchors.

Figure 1

DNA-AuNP probes constructed by conjugating oligonucleotides having different organosulfur anchor groups to AuNPs. SP-1, SP-2, and SP-3 represent DNA-AuNP probes that involve conjugation through thiol, acyclic disulfide, and cyclic disulfide (DTPA), respectively.

Figure 2

Thermal-induced release of FAM-labeled DNA from SP-1. (A) Schematic illustrating the fluorescence-based measurement of the released FAM-labeled DNA from DNA-AuNP. (B) Fraction of released DNA from SP-1 over a period of 20 h at different temperatures (25, 37, 60, and 85 °C). (C) Determination of the rate constants k based on the plots. (D) The logarithmic value of rate constants as a function of reciprocal of temperatures, providing information on the activation energy of the process. Error bars represent one standard deviation from triplicate sample analyses.

Figure 3

Fraction of FAM-labeled DNA released from the three DNA-AuNPs probes (SP-1, SP-2, and SP-3) over a period of 20 h at 37 °C (A) and at 85 °C (B). Error bars represent one standard deviation from triplicate sample analyses. Inset photographs in B were taken after 20 h of incubation at 85 °C, showing colors of different sample solutions at this time point.

Figure 4

Release of FAM-labeled DNA from the DNA-AuNPs when the DNA-AuNPs were treated with 50 μM mercaptohexanol (MCH) at 25 °C. (A) Schematic illustrating the fluorescence-based measurement of the chemical release of FAM-labeled DNA from the DNA-AuNPs. (B) Fraction of FAM-labeled DNA released from DNA-AuNPs having three different anchor groups (SP-1, thiol; SP-2, acyclic disulfide; and SP-3, DTPA).

Figure 5

Effect of packing density on the thermal stability of DNA-AuNPs at 37 °C (A) and at 85 °C (B). The average density ranged from 90 oligonucleotide molecules per AuNP to 190 oligonucleotide molecules per AuNP. Error bars represent one standard deviation from triplicate sample analyses.

Figure 6

Design of double-stranded DNA-AuNP probes to enhance the stability of the short internal complementary DNA (sicDNA). (A) Schematic showing the double-stranded DNA-AuNP probes constructed by conjugating both of the complementary strands with different combinations of organosulfur anchors. DP-1 represents DNA-AuNP constructed by conjugating both DNA strands with thiol anchors; DP-2 represents DNA-AuNP by conjugating one DNA strand with the thiol anchor and the other strand with the DTPA anchor; DP-3 represents DNA-AuNP by conjugating DNA strands with DTPA anchors. (B) Release of DNA from DNA-AuNPs induced by 2 mM DTT at room temperature. (C) Release of DNA from double-stranded DNA-AuNP probes at a temperature of 37 °C, which is lower than the melting temperature (48.6 °C) of the double-stranded DNA (dsDNA). (D) Comparison of the thermal stability of DP-1 (two thiol anchors) with its control (a single thiol anchor) at a temperature of 60 °C, which is higher than the melting temperature (48.6 °C) of the dsDNA. Error bars represent one standard deviation from triplicate sample analyses.