Protein replacement therapy is a powerful and versatile approach for treating disease because it can be used to replace dysregulated proteins in cells or manipulate cellular signaling. However, transfecting – or deliberately introducing – protein-based materials into cells where they can perform such tasks is exceedingly difficult. For one thing, cells are very good at protecting themselves and keeping unwanted foreign entities out, and many proteins are quickly degraded in the cellular environment. Furthermore, the sizes, charges, and hydrophobicities of proteins inherently hinder their uptake. Therefore, generalizable methods to ‘package’ proteins that allow them to be efficiently transfected are needed.

In 1996, our group introduced a new class of nanostructure, consisting of a gold nanoparticle surface-functionalized with a dense and radially oriented DNA shell. Structurally, these spherical nucleic acids, or SNAs, can be thought of as a nanoscale version of a ‘koosh ball’. ‘Koosh balls’ have a rigid steel-bound core with rubber filaments radiating out from it. Over the last 25 years, our group and others have synthesized dozens of different versions of SNAs with a variety of core and nucleic acid shell compositions. Indeed, one of the powerful features of this architecture is that it is highly modular, and the cores and shells can be swapped out independently; the properties of such conjugate materials are a synergistic combination of those of the core and the shell. So, in the case of the SNA, even if the core is changed, as long as the shell is the same, the conjugate retains many of its defining features owing to the three-dimensional arrangement of nucleic acids in the ligand shell.

In 2006, our lab showed for the first time that SNAs with gold cores easily enter cells in high quantities, and we and others have since used them to open new areas in sensing, diagnostics, and therapeutics. For these applications, we recognized that biocompatible SNAs, which avoided the use of inorganic or metallic core materials that might be incompatible with biological systems, would be advantageous. And so, a new class of SNA with a protein core - protein spherical nucleic acids, or ProSNAs (in addition to a variety of other ones with hollow, micelle, liposomal, or polymeric cores) was synthesized and advanced. In simple terms, the gold nanoparticle core was exchanged for a protein core, leaving the DNA shell the same. As expected, ProSNAs retain many of the properties of the prototypical SNAs that make them useful in biology and medicine. Importantly, ProSNAs can be thought of as a non-toxic, nucleic acid-wrapped protein ‘package’ capable of being transfected. Many different types of protein cores and nucleic shells can be used to prepare ProSNA structures, enabling tailorable uptake and targeted diagnostic and therapeutic approaches (see Figure).

Figure created in Biorender.com

In this Nature Protocol article, we systematically describe the synthesis of ProSNAs through the DNA modification of accessible chemical modalities on the surface of proteins. First, amino acid sites on the protein surface are chemically modified to enable DNA attachment. After purification, DNA strands are then added in excess to make the protein-DNA conjugate. ProSNAs exhibit increased cellular uptake compared to an unmodified (or native) protein, and we describe how they can be used as intracellular probes for glucose detection and functional protein delivery in whole organisms (in vivo), as representative examples. Thus, this protocol highlights an important strategy for protein transfection but also novel approaches to detection and therapeutics more broadly that will be very useful to the scientific community.

Proteins have been studied for over two centuries, but we are still discovering new and revolutionary ways to harness their biological potential, especially at the nanoscale. In this protocol, we show that pairing them with DNA enhances their intracellular delivery, opening a new swath of possibilities in the fundamental and applied realms.

SHP & JHO contributed significantly to the writing of this post.