Targeted and reversible cancer cell-binding DNA nanoparticles

Abstract

DNA nanoparticles (DeNAno) produced by rolling circle replication of circular oligonucleotide templates are a novel format for the selection of cell-binding reagents from randomized libraries. DeNAno particles consist of several hundred concatemers and can leverage that multivalency to bind to complex surfaces such as cells. In this study, an iterative bio-panning approach was used to recover particles that bound to the mouse pancreatic cancer line, Panc-02. These particles shared a primary sequence motif. Hybridization of a locked nucleic acid complimentary to this motif both inhibited cell binding and released pre-bound DeNAno particles from the cells. The monomeric form of one of the selected sequences was unable to compete with the cognate particle, consistent with a low-affinity, but high-avidity, type interaction. DeNAno library selection against cancer or other cell types can, thus, yield novel cell binding agents without a priori knowledge of particular cell surface molecules.

Selection of DNA nanoparticles with preferential binding to aggregated protein target

“…For TEM, both ‘standard’ (the size of particle used throughout the paper) and ‘small’ (used in Figure 2D ) particles were made. In all cases, discreet ‘balls’ of DNA were observed, with a diameter of 100–250 nm (AFM), 75 nm (standard DeNAno/TEM) or, 58 nm (small DeNAno/TEM), similar in size to previous reports using nanoparticle tracking system (Nanosight) ( 2 ). The disparity between TEM and AFM size of standard DeNAno is most likely due to the compactness of the imaged DeNAno, which is partly determined by the amount of salt present in the sample and the preparation method.…”

“…DeNAno particles have previously been selected against cellular targets ( 1 , 2 ). However, characterization of these particles has been limited by the anonymity of the target.…”

“…DeNAno DNA particles are a novel multivalent reagent that relies on high overall avidity instead of high affinity to bind their targets. DeNAno particles that specifically bind to primary human dendritic cells ( 1 ) and the mouse pancreatic cancer cell line Panc-02 ( 2 ) have been selected previously. The selection process is a biopanning strategy akin to that used in aptamer selection by systemic evolution of ligands by exponential enrichment (SELEX), in which a highly diverse library of DNA particles is incubated with the target to capture binders followed by amplification and iteration of the process.…”

“…The selection process is a biopanning strategy akin to that used in aptamer selection by systemic evolution of ligands by exponential enrichment (SELEX), in which a highly diverse library of DNA particles is incubated with the target to capture binders followed by amplification and iteration of the process. While aptamers are generally small pieces of DNA or RNA (<100 bp) that bind in a monovalent fashion with high affinity, DeNAno are concatemers of up to several hundred copies in length made by rolling circle amplification (RCA), with sizes that can be several hundred nanometers ( 2 ). This long strand of DNA forms secondary and tertiary structure, which is the basis for ability to bind their targets specifically.…”

“…The template strand is enriched via asymmetric polymerase chain reaction (PCR), circularized and the selection process is repeated until binding particles dominate the pool. As with aptamers, DeNAno with primary sequence motifs have been identified ( 2 ).…”