The Oligonucleotide Nanoparticle Conjugate and the "Antisense Nanoparticle"

Speaker:
Chad Mirkin, Northwestern University

Highlights

• Oligonucleotides tethered to gold nanoparticles form a new entity with promise for diagnostics and therapeutics.
• Choosing the sequences on the nanoparticles and the complementary sequences on molecules that link them can promote self-assembly into the desired crystal structures.
• Complementary oligonucleotides pair cooperatively when bound to gold, enhancing both their sensitivity and selectivity.
• Oligonucleotide-covered nanoparticles rapidly enter a variety of cells, so they could be useful for delivering antisense DNA or other nucleic acid payloads.
• A single "nanoflare" particle both binds RNA and reports the binding through fluorescence.

Programming interactions with oligonucleotides

Chad Mirkin and his group at Northwestern University are pursuing a variety of approaches to control materials on a 1–100nm length scale. Their well known "dip-pen lithography" technique, for example, uses scanning-probe microscopes to spatially place molecules, including those, like proteins or oligonucleotides, that are too fragile for conventional lithgraphic methods. Recently they have extended this method from a single tip to as many as 55,000 cantilevers in parallel to create patterns over areas large enough to be measured in square-centimeters. They are also exploring supramolecular assembly and anisotropic nanostructures.

In his talk at Hunter, Mirkin focused on nanoparticles decorated with oligonucleotides. The promising and surprising properties of this combination earns it a unique new name, he said, opting for "oligonucleotide–nanoparticle conjugate." However awkward this nomenclature, these particles could be a powerful tool in both diagnostic and therapeutic bionanotechnology.

"Our view was that we would have to rely on inspiration from biology."

The researchers first developed these particles for biomolecule-directed assembly, exploiting the ability to routinely synthesize arbitrary nucleotide sequences. Base pairing between complementary sequences then provides the detailed assembly instructions referred to by Matsui. "Our view was, in the mid-90s, that we would have to rely on biological systems, or at least inspiration from biology."

Most of their work has used gold particles, Mirkin said, although the team has explored other noble metals as well as semiconductors, insulators, and magnetic nanoparticles. Gold has an advantage because it is usually prepared using a technique that leaves it covered in a shell of weakly bound ligands, so that oligonucleotides linked to thiols can displace them in large numbers—as many as 200 per particle.

In recent work, Mirkin and his colleagues have chosen sequences that promote the formation of crystallites with either a face-centered- (fcc) or body-centered-cubic (bcc) crystal structure. The researchers used the same oligonucleotide-decorated spheres in both cases, but changed the sequences used to bind them together. For the fcc structure, a single strand links the spheres, encouraging close packing, while for the bcc structure, two distinct strands make the connection.

Greater than the sum of the parts

Even when the nanoparticles don't form a regular crystal, their interaction can be seen "with the naked eye," Mirkin obvserved. Close proximity shifts the plasmon resonance of the gold spheres, changing the color from red to what Mirkin called "Northwestern purple." This color change sensitively monitors the binding of complementary oligonucleotides.

Densely attaching oligonucleotides to a gold nanoparticle creates a new entity with promise for both diagnostics and therapeutics.

A critical—and unexpected—observation was that oligonucleotides pair much more suddenly when they are densely attached to spheres than when they are floating freely. Instead of requiring a temperature decrease of about 25 degrees to get complete pairing, the transition for the nanoparticles system occurs in a single degree.

"We get particles that have properties that are very different from the inorganic core and the oligonucleotides from which they derive," Mirkin said. "That's one of the names of the game in nanoscience." One reason for the sharp transition is that there are multiple links between spheres. In addition, he suggested, the highly charged oligonucleotides attract oppositely charged counter ions which help to stabilize other oligonucleotides nearby. "It's a combination of these two effects that leads to these very narrow transitions."

In diagnostic assays, the sharper transition improves the selectivity of the assay, Mirkin said, to the extent that the binding change from a single point mutation can be detected. The narrower transition also improves the sensitivity, he observed, since weakly bound species completely unbind with no loss of the target species. A commercial tool based on this research uses the catalysis of silver reduction by the gold nanoparticles to achieve a 100,000-fold increase in sensitivity.

Delivering the goods

Although oligonucleotide–nanoparticle conjugates have great promise in these diagnostic applications, Mirkin spoke even more enthusiastically about their potential therapeutic use. In this context, the particles act in some ways like a customizable antisense RNA, but with important advantages, including tighter binding to their target because of the cooperativity.

The first challenge is to see whether the particles enter cells at all. Many other carriers have been used for this purpose, and "the lore in the literature is that you need positively charged entities," Mirkin said. In contrast, these conjugates are "some of the most negatively charged materials you can get," because of the oligonucleotides on the surface.

"We have yet to find a cell line where we don't get greater than 99.9% transfection."

Nonetheless, the particle complexes are very effectively taken up by a wide variety of cell lines. "We have yet to find a cell line where we don't get greater than 99.9% transfection," Mirkin said, "including nerve cells." They enter by endocytosis, he observed, but nonetheless retain their discrete character rather than being digested. Their resistance to degradation by nucleases may reflect their steric inaccessibility on the nanoparticle, Mirkin suggested. "That should translate into higher activity."

The uptake appears to be assisted by specific extracellular proteins, which tend to partially cancel the charge, the researchers found. Identifying these proteins could even avoid the need for the nanoparticles, Mirkin said, since they might act as a universal transfection agent when attached to "whatever it is you want to carry into the cell."

Mirkin's team confirmed that the nanoparticles can bind to messenger RNA in cells to reduce expression of green fluorescent protein in vitro. "We get about 60% knockdown, which is about as good as you can do under conditions where we have a lot of cell division," he said. Thus the oligonucleotide-nanoparticle conjugate shows great potential for delivering nucleic acids into cells.

Combining functions

The binding to complementary RNA can be made stronger, Mirkin noted, by using "locked nucleic acid," or LNA, chains, in place of the DNA oligonucleotides. (LNA is a bicyclic high affinity RNA analog in which the ribofuranose ring is locked in the 3′-endo conformation.) Team member Dwight Seferos used these LNA-modified particles to persistently knock down the activity of the survivin gene, which Mirkin said makes lung-cancer cells "effectively immortal."

Finally, Mirkin illustrated steps toward combining diagnostic and therapeutic activity in a single, multifunctional particle. His team created what they call a "nanoflare," in which a short complementary sequence weakly binds a fluorophore close to the gold nanoparticle, which quenches its fluorescence. When the longer target sequence binds to the complementary oligonucleotide, it also displaces the fluorophore, allowing it to emit light. "The beauty of this," Mirkin said, is that researchers can use nanotechnology to "build one kind of structure that gives you all of those capabilities."