Nuclease-resistant DNA via high-density packing in polymeric micellar nanoparticle coronas

Abstract

Herein, we describe a polymeric micellar nanoparticle capable of rendering nucleic acids resistant to nuclease digestion. This approach relies on utilizing DNA as the polar headgroup of a DNA-polymer amphiphile in order to assemble well-defined, discrete nanoparticles. Dense packing of DNA in the micelle corona allows for hybridization of complementary oligonucleotides while prohibiting enzymatic degradation. We demonstrate the preparation, purification, and characterization of the nanoparticles, then describe their resistance to treatment with endo- and exonucleases including snake-venom phosphodiesterase (SVP), a common, general DNA digestion enzyme.

Figure 1

Preparation of DNA-polymer amphiphiles (DPAs) and assembly of micelles. Synthesis: i) A hydrophobic polymer, terminally modified with a carboxylic acid moiety was mixed with a coupling agent and reacted with a 5′-amino modified oligonucleotide on solid support (controlled pore glass, CPG). ii) Deprotection and cleavage of the resulting DNA-polymer conjugate from solid support. iii) Dialysis of cleaved DPA into deionized water to form a mixture of micelles and free, non-conjugated nucleic acid. T_F and T_D correspond to fluorescein- and DABCYL-modified thymidine phosphoramidites. a) PAGE analysis. Lane 1: Crude material post-micelle (P1) formation showing conjugate (top band) and free ssD-NA (lower band). Lane 2: HPLC purified sample of ssDNA-1. Lane 3: Purified P1, isolated via size-exclusion chromatrography (SEC). b) SEC trace of purified P1 (λ_abs = 260 nm). c) Transmission electron micrograph of P1. See Supporting Information Figure S7 for P2 data.

Figure 2

Endonuclease resistance of DPA nanoparticles. a) Scheme depicting DPA-nanoparticle (P2) resistance to nicking endonuclease (Nt.CviPII) and consequently intact, quenched duplex DNA at the particle surface. b) Scheme depicting dsDNA degradation by Nt.CviPII and consequently a decrease in duplex melting temperature and increase in fluorescein fluorescence. c) Nt.CviPII activity over time, monitored via fluorescein fluorescence dequenching (λ_ex = 485 nm, λ_em = 535 nm). d) Thermal denaturation analysis with and without Nt.CviPII treatment for P1 and ssDNA-1. λ_abs = 260 nm. Sample subjected to enzyme for 100 minutes at 37 °C. ssDNA-1 + Complement: T_m = 63.9 °C; ssDNA-1 + Nt.CviPII + Complement: T_m = 37.8 °C; P1 + Complement: T_m = 58.8 °C; P1 + Nt.CviPII + Complement: T_m = 58.3 °C). e) Thermal denaturation analysis with and without Nt.CviPII treatment for P2 and ssDNA-2. λ_abs = 260 nm. Sample subjected to enzyme for 100 minutes at 37 °C. ssDNA-2 + Complement: T_m = 63.9 °C; ssDNA-2 + Nt.CviPII + Complement: T_m = 37.8 °C; P2 + Complement: T_m = 56.9 °C; P2 + Nt.CviPII + Complement: T_m = 55.3 °C). See Supporting Information Figure S12 for derivative plots of melting temperatures. Complement: 5′-TATTATATCTTTAGACACTGA CTGGACATGACTCT-3′

Figure 3

Exonuclease resistance of DPA nanoparticles. a) Scheme depicting DPA-nanoparticle resistance to Exonuclease III and consequently, intact DNA at the particle surface available for hybridization with complementary ssDNA. b) Scheme depicting ssDNA being degraded by ExoIII and consequently, no intact DNA available for hybridization with complementary ssDNA. c) Exonuclease III activity over time monitored by fluorescein fluorescence dequenching (λ_ex = 485 nm, λ_em = 535 nm). d) Thermal denaturation analysis with and without Exo III treatment for P1 and ssDNA-1. λ_abs = 260 nm. Samples subjected to enzyme for 60 minutes at 37 °C. P1 + Complement: T_m = 58.8 °C; P1 + Exo III + Complement: T_m = 55.8 °C). e) Thermal denaturation analysis with and without Exo III treatment for P2 and ssDNA-2. λ_abs = 260 nm. Samples subjected to enzyme for 60 minutes at 37 °C. P2 + Complement: T_m = 56.9 °C; P2 + Exo III + Complement: T_m = 53.9 °C). See Supporting Information Figure S13 for derivative plots of melting temperatures. Complement: 5′-TATTATATCTTTAGACAC TGACTGGACATGACTCT-3′

Figure 4

SVP activity over time monitored by fluorescein fluorescence dequenching (λ_ex = 485 nm, λ_em = 535 nm).