Cooperative effects in DNA-functionalized polymeric nanoparticles

Abstract

DNA-functionalized nanoparticles (NPs), called spherical nucleic acids (SNAs), have attracted considerable attention due to their unique properties and numerous applications. In particular, DNA-functionalized dye-loaded polymeric NPs (DNA-NPs), owing to their exceptional fluorescence brightness, have emerged as powerful nanomaterials for the ultrasensitive detection and imaging of nucleic acids. Herein, we addressed a fundamental question unexplored for polymeric DNA-NPs: how does the dense packing of oligonucleotides on the particle surface impact their capacity to specifically hybridize with complementary sequences? Using Förster resonance energy transfer (FRET) between DNA-NPs and labelled complementary strands, we found that the DNA on the surface of the NPs exhibits dramatic enhancement in duplex stability compared to free DNA duplexes (>20 °C). This effect increases at higher densities of coding DNA on the NP surface, which suggests that DNA cooperativity is responsible for the enhancement in duplex stability. For example, 8 nt DNA duplexes were perfectly stable at RT on the surface of DNA-NPs. Furthermore, these DNA-NPs preserve the capacity to distinguish mutations, even at the single-nucleotide level within a 21 nt sequence, when an appropriate hybridization temperature is used. The hybridization between DNA-NPs and the complementary sequences proceeds on the min time scale at probe and target concentrations of ≥10 and ≥100 pM, respectively. Below these, this diffusion-controlled process becomes too slow, indicating the fundamental limitation in DNA/RNA sensing assays that require sufficiently high nanoprobe concentration. The present study sheds light on the capacity of DNA-NPs to specifically hybridize with the target sequences and provides insights into the development of nucleic acid sensing assays.

Fig. 1. Chemical structures of the PEMA-AspN3 polymer and dye R18/F5-TPB, their nanoprecipitation into bare dye-loaded polymeric NPs with functional azide groups, functionalization of the latter into DNA-NPs and hybridization of the complementary stands with a FRET acceptor.

Fig. 2. TEM image (left) and size distribution diagram (right) of PEMA-AspN3 NPs functionalized at 100% with a survivin capture coding sequence and loaded with 33 wt% of R18/F5-TPB dye (with respect to total NP mass). Scale bar: 50 μm.

Fig. 3. DNA hybridization in DNA-NPs with varying surface density of coding sequences studied by FRET. (A) Fluorescence spectra of 100% coding (100% survivin capture sequence) and 10% coding (10% survivin capture sequence, 90% A20 sequence) DNA-NPs (50 wt% of R18/F5-TPB dye with respect to the polymer, meaning 33 wt% of the total mass of the NPs) hybridized at 40 °C for 20 min, with varying concentrations of acceptor dye bearing a DNA sequence of 21 nucleotides, representing part of the survivin target, thus being fully complementary to the capture sequence on the DNA-NPs. For this experiment, 100 pM of DNA-NPs were hybridized with 0.5–50 nM of the acceptor-sequence. Negative control: DNA-NPs without the addition of the acceptor-sequence. Mg buffer was systematically used. Spectra were measured at 530 nm excitation at RT. (B) FRET ratio for the 100% coding and 10% coding DNA-NPs, measured by varying the concentration of the acceptor-sequence.

Fig. 4. Thermal stability of the duplexes. (A) Schematic representation of the single-stranded donor and acceptor-sequence pairs and the NP and acceptor-sequence pairs. The oligonucleotide sequences attached to the donor and acceptor are presented. (B) FRET ratios for increasing temperature for the pairs of a single dye donor: 8-nt acceptor (A8, in black) and 12-nt acceptor (A12, in red). In this experiment, 3.3 nM of the donor-sequence was hybridized with 10 nM of the acceptor-sequence. (C) Fluorescence spectra of 100% coding DNA-NPs hybridized with the 8-nt acceptor and subjected to increasing temperatures from 20 °C to 60 °C. (D) FRET ratios for 10% and 100% coding DNA-NPs, hybridized with the 8-nt and 12-nt acceptors. In this experiment, 100 pM of DNA-NPs was hybridized with 10 nM of the acceptor-sequence. The temperature was increased from 20 °C to 60 °C (for 10% coding DNA-NPs) or to 80 °C (for 100% coding DNA-NPs). Mg buffer was systematically used. Spectra were measured at 530 nm excitation.

Fig. 5. Reversibility of FRET in heating–cooling cycles for DNA-NPs (100 pM), 100% coding with the survivin capture sequence in the presence of the 8-nt acceptor (10 nM): emission spectra (A) and corresponding FRET ratios (B). Spectra were measured at 530 nm excitation.

Fig. 6. Sensitivity to mutations. (A) Schematic representation of the NP hybridized with acceptor-sequences each containing zero (A-21), one (A-21_MUT1) or three (A-21_MUT3) point mutations, as indicated by the yellow nucleotides. (B) Normalized fluorescence spectra for 10% coding DNA-NPs hybridized with acceptor sequences with zero (in black), one (in red) or three (in blue) point mutations at three different temperatures of 40 °C, 60 °C and 70 °C. (C) FRET ratios vs. tempetature for 10% and 100% coding DNA-NPs, hybridized with acceptor-sequences with zero, one or three point mutations. In these experiments, 100 pM of DNA-NPs was hybridized with 10 nM of the acceptor-sequence. The temperature was increased from 20 °C to 70 °C. Mg buffer was systematically used. Spectra were measured at 530 nm excitation.

Fig. 7. Kinetics of hybridization. (A) Normalized (and smoothed) fluorescence spectra for 100% coding DNA-NPs mixed with the acceptor-sequence with 21 nucleotides at a ratio of 1 NP : 10 21-nt acceptor in low (1 pM DNA-NPs) and high (37.5 pM DNA-NPs) concentrations. The spectra were recorded immediately after mixing for a duration of 10 minutes at the frequency of one spectrum per minute. Schematic representation of the DNA-NPs and acceptor-sequences in solution at different concentrations. (B) FRET ratios for varying concentrations of 100% coding DNA-NPs: 21-nt acceptor up to 10 minutes. The curves connecting the data points are the polynomial fits. In this experiment, the ratio of 1 NP :10 21-nt acceptor was maintained throughout, starting with 1 pM of DNA-NPs hybridized with 10 pM of the 21-nt acceptor and increasing the concentration. Mg buffer was systematically used. Spectra were measured at 530 nm excitation.