What controls the hybridization thermodynamics of spherical nucleic acids?

Abstract

The hybridization of free oligonucleotides to densely packed, oriented arrays of DNA modifying the surfaces of spherical nucleic acid (SNA)-gold nanoparticle conjugates occurs with negative cooperativity; i.e., each binding event destabilizes subsequent binding events. DNA hybridization is thus an ever-changing function of the number of strands already hybridized to the particle. Thermodynamic quantification of this behavior reveals a 3 orders of magnitude decrease in the binding constant for the capture of a free oligonucleotide by an SNA conjugate as the fraction of pre-hybridized strands increases from 0 to ∼30%. Increasing the number of pre-hybridized strands imparts an increasing enthalpic penalty to hybridization that makes binding more difficult, while simultaneously decreasing the entropic penalty to hybridization, which makes binding more favorable. Hybridization of free DNA to an SNA is thus governed by both an electrostatic barrier as the SNA accumulates charge with additional binding events and an effect consistent with allostery, where hybridization at certain sites on an SNA modify the binding affinity at a distal site through conformational changes to the remaining single strands. Leveraging these insights allows for the design of conjugates that hybridize free strands with significantly higher efficiencies, some of which approach 100%.

Figure 1

(a) Quantification of the fraction of strands that hybridize to an SNA functionalized with ∼80 complementary strands as a function of stoichiometry for oligonucleotides of several different lengths. (b) Dehybridization “melting” curves for SNAs prepared with differing numbers of hybridized 9-mer complementary strands, enumerated in the legend. Inset: the first derivative of each curve, the full width at half-maximum of which is a measure of the “sharpness” of the transition. (c) Calculated SNA binding constants as a function of the number of pre-hybridized strands. Inset: the linear van't Hoff plots from which the thermodynamic quantities have been extracted. The legend describes the values of n probed in this experiment. (d) Trends in the enthalpy (left axis, blue) and entropy (right axis, green) of SNA binding as a function of the number of pre-hybridized strands.

Figure 2

(a) Quantification of the fraction of strands that hybridize to an SNA as a function of increased bulk salt concentration. The dashed line represents the salt concentration used in prior experiments. (b) Probability distribution function (PDF) from molecular dynamics simulations indicates that the radius of gyration of unhybridized DNA on the SNA elongates as more sites on the SNA are duplexed. Inset: the change in ΔS plotted over the entire range of binding site duplexation (0–80 strands), using the PDF fits and Figure 1d. The experimental and model data are fit with a scaling exponent γ = 0.55 (green line) that predicts the behavior of both the experimental and MD simulation data.

Figure 3

(a) Comparison of binding isotherms obtained from SNAs functionalized with either ∼40 or ∼80 DNA strands. Insets: schematic illustration of the role of oligo ethylene glycol moieties (shown in green) in diluting the surface DNA density compared to the densely functionalized particles investigated previously. (b) Comparison of the binding constants of SNAs functionalized with ∼40 or ∼80 surface-bound DNA strands and prehybridized with n = 20 duplexes.

Scheme 1

Pre-hybridization of Spherical Nucleic Acid–Gold Nanoparticle Conjugates with n Strongly Bound Oligonucleotides (Blue) To Allow Independent Measurement of the Binding Constant of the n+1 Strand (Yellow)