DeNAno: Selectable deoxyribonucleic acid nanoparticle libraries

Abstract

DNA nanoparticles of approximately 250 nm were produced by rolling circle replication of circular oligonucleotide templates which results in highly condensed DNA particulates presenting concatemeric sequence repeats. Using templates containing randomized sequences, high diversity libraries of particles were produced. A biopanning method that iteratively screens for binding and uses PCR to recover selected particles was developed. The initial application of this technique was the selection of particles that bound to human dendritic cells (DCs). Following nine rounds of selection the population of particles was enriched for particles that bound DCs, and individual binding clones were isolated and confirmed by flow cytometry and microscopy. This process, which we have termed DeNAno, represents a novel library technology akin to aptamer and phage display, but unique in that the selected moiety is a multivalent nanoparticle whose activity is intrinsic to its sequence. Cell targeted DNA nanoparticles may have applications in cell imaging, cell sorting, and cancer therapy.

Figure 1

Production and basic characterization of DNA nanoparticles. a) DNA nanoparticles are produced by circularizing a 100nM concentration of a 94 base ssDNA template with T4 Ligase and a 300nM concentration of a 31 base templating primer. Polymerization was done with phi29 DNA polymerase at 30°C for 30 minutes and terminated with EDTA. Discrete particles are stained with SYBR Green and viewed under a 100X oil objective. b) Nanoparticles created for various reaction times are measured with Dynamic Light Scattering to validate size and demonstrate positive correlation of hydrodynamic radius with reaction time

Figure 2

DNA nanoparticle iterative selection scheme. ssDNA libraries are ligated with T4 ligase and polymerized with phi29 DNA polymerase. 3'–5' exonuclease activity of phi29 DNA polymerase ensures nanoparticle purity from extraneous DNA. Immature DCs were cultured in RPMI 1640 medium supplemented with 2 mM L-glutamine, 50 mM 2-mercaptoethanol, 10 mM HEPES, penicillin (100 U/mL), streptomycin (100mg/mL), 5% human AB serum, 1000 U GM-CSF/mL and 200U IL-4/mL and harvested in days 5–7. Cell incubation and washing followed by QPCR (200nM primers, 95°C 2min, cycle 95°C 30sec, 61°C 1min, 72°C 20sec to completion. 5μL of resultant reaction was added to 45 μL fresh PCR buffer with 400nM phosphorlyated template primer v6F. 10 additional cycles of PCR generate an excess of the desired single strand. DNA was purified with a QIAquick Nucleotide Removal Kit (Qiagen, Valencia, CA), eluted into T4 DNA Ligase Buffer and recircularized to begin the next round. Nine rounds were produced after which sequences were cloned using a pGEM-T cloning kit (Promega, Madison, WI).

Figure 3

Selection of dendritic cell binding DNA nanoparticles. (a) Nine rounds of selection were performed, after which the selected population was labeled by incorporation of fluorescent nucleotides and the binding to dendritic cells evaluated by flow cytometry. A random clone from the library was used as a negative control. (b) From the ninth round of selection, individual population members were cloned, sequenced and regenerated with fluorescent nucleotides. The incorporation efficiency of Alexa488 OBEA-dCTPs by phi29 polymerase was calculated to be ~1.5%. Controls include an irrelevant DNA nanoparticle and the reaction mix containing the fluorescent dNTPs.

Figure 4

DC specific binding by clone 3 DNA nanoparticle. Clone 3 particles were generated with incorporated fluorescent nucleotides and evaluated for binding to DCs as well as P815 and THP1 cell lines by flow cytometry and fluorescent microscopy. For each flow cytometry plot (shown on the left), the cells with the labeled clone 3 particles are shown in green, a control particle that is made from the complementary sequence of clone 3 is shown in blue, and the cells alone are indicated by the red curve. Microscopy images show bright field, fluorescent and overlays (from left to right) of fixed cells incubated on ice with the labeled clone 3 particles. Cells were washed 3 times before imaging. Fluorescent staining was seen only on the DC. The complementary control particles did not produce any fluorescent labeling (data not shown).