skip to main content


Title: Trapping of protein cargo molecules inside DNA origami nanocages
The development of the DNA origami technique has directly inspired the idea of using three-dimensional DNA cages for the encapsulation and targeted delivery of drug or cargo molecules. The cages would be filled with molecules that would be released at a site of interest upon cage opening triggered by an external stimulus. Though different cage variants have been developed, efficient loading of DNA cages with freely-diffusing cargo molecules that are not attached to the DNA nanostructure and their efficient retention within the cages has not been presented. Here we address these challenges using DNA origami nanotubes formed by a double-layer of DNA helices that can be sealed with tight DNA lids at their ends. In a first step we attach DNA-conjugated cargo proteins to complementary target strands inside the DNA tubes. After tube sealing, the cargo molecules are released inside the cavity using toehold-mediated strand displacement by externally added invader strands. We show that DNA invaders are rapidly entering the cages through their DNA walls. Retention of ∼70 kDa protein cargo molecules inside the cages was, however, poor. Guided by coarse-grained simulations of the DNA cage dynamics, a tighter sealing of the DNA tubes was developed which greatly reduced the undesired escape of cargo proteins. These improved DNA nanocages allow for efficient encapsulation of medium-sized cargo molecules while remaining accessible to small molecules that can be used to trigger reactions, including a controlled release of the cargo via nanocage opening.  more » « less
Award ID(s):
1827346
NSF-PAR ID:
10387690
Author(s) / Creator(s):
; ; ; ; ; ; ;
Date Published:
Journal Name:
Nanoscale
Volume:
14
Issue:
48
ISSN:
2040-3364
Page Range / eLocation ID:
18041 to 18050
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. null (Ed.)
    Nature utilizes self-assembled protein-based structures as subcellular compartments in prokaryotes to sequester catalysts for specialized biochemical reactions. These protein cage structures provide unique isolated environments for the encapsulated enzymes. Understanding these systems is useful in the bioinspired design of synthetic catalytic organelle-like nanomaterials. The DNA binding protein from starved cells (Dps), isolated from Sulfolobus solfataricus , is a 9 nm dodecameric protein cage making it the smallest known naturally occurring protein cage. It is naturally over-expressed in response to oxidative stress. The small size, natural biodistribution to the kidney, and ability to cross the glomerular filtration barrier in in vivo experiments highlight its potential as a synthetic antioxidant. Cytochrome C (CytC) is a small heme protein with peroxidase-like activity involved in the electron transport chain and also plays a critical role in cellular apoptosis. Here we report the encapsulation of CytC inside the 5 nm interior cavity of Dps and demonstrate the catalytic activity of the resultant Dps nanocage with enhanced antioxidant behavior. The small cavity can accommodate a single CytC and this was achieved through self-assembly of chimeric cages comprising Dps subunits and a Dps subunit to which the CytC was fused. For selective isolation of CytC containing Dps cages, we utilized engineered polyhistidine tag present only on the enzyme fused Dps subunits (6His-Dps-CytC). The catalytic activity of encapsulated CytC was studied using guaiacol and 3,3′,5,5′-tetramethylbenzidine (TMB) as two different peroxidase substrates and compared to the free (unencapsulated) CytC activity. The encapsulated CytC showed better pH dependent catalytic activity compared to free enzyme and provides a proof-of-concept model to engineer these small protein cages for their potential as catalytic nanoreactors. 
    more » « less
  2. Abstract

    We characterize the encapsulation of supercharged green fluorescent protein, GFP(+36), by thermophilic ferritin fromArchaeoglobus fulgidus(AfFtn). The AfFtn–GFP(+36) assembly is rapid, nearly stoichiometric, and robust. Using a more stably assembled mutant AfFtn, we show that encapsulation can occur in the presence of mostly assembled cages, in addition to encapsulation starting from AfFtn individual subunits. Assembly and encapsulation do not occur with non‐supercharged GFP or the alternately supercharged GFP(−30), highlighting the role of complementary electrostatic interactions between the cargo and AfFtn cage interior. We also present a method for verifying protein–protein encapsulation, using nickel nitrilotriacetic acid agarose resin. AfFtn‐supercharged protein host‐guest complexes could find applications in enzyme studies, protein separations, andin vivoprotein stabilization and targeted delivery.

     
    more » « less
  3. Neonatal hypoxic-ischemic encephalopathy is the leading cause of permanent brain injury in term newborns and currently has no cure. Catalase, an antioxidant enzyme, is a promising therapeutic due to its ability to scavenge toxic reactive oxygen species and improve tissue oxygen status. However, upon in vivo administration, catalase is subject to a short half-life, rapid proteolytic degradation, immunogenicity, and an inability to penetrate the brain. Polymeric nanoparticles can improve pharmacokinetic properties of therapeutic cargo, although encapsulation of large proteins has been challenging. In this paper, we investigated hydrophobic ion pairing as a technique for increasing the hydrophobicity of catalase and driving its subsequent loading into a poly(lactic-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) nanoparticle. We found improved formation of catalase-hydrophobic ion complexes with dextran sulfate (DS) compared to sodium dodecyl sulfate (SDS) or taurocholic acid (TA). Molecular dynamics simulations in a model system demonstrated retention of native protein structure after complexation with DS, but not SDS or TA. Using DS-catalase complexes, we developed catalase-loaded PLGA-PEG nanoparticles and evaluated their efficacy in the Vannucci model of unilateral hypoxic-ischemic brain injury in postnatal day 10 rats. Catalase-loaded nanoparticles retained enzymatic activity for at least 24 h in serum-like conditions, distributed through injured brain tissue, and delivered a significant neuroprotective effect compared to saline and blank nanoparticle controls. These results encourage further investigation of catalase and PLGA-PEG nanoparticle-mediated drug delivery for the treatment of neonatal brain injury. View Full-Text 
    more » « less
  4. Abstract

    The use of DNA‐based nanomaterials in biomedical applications is continuing to grow, yet more emphasis is being put on the need for guaranteed structural stability of DNA nanostructures in physiological conditions. Various methods have been developed to stabilize DNA origami against low concentrations of divalent cations and the presence of nucleases. However, existing strategies typically require the complete encapsulation of nanostructures, which makes accessing the encased DNA strands difficult, or chemical modification, such as covalent crosslinking of DNA strands. We present a stabilization method involving the synthesis of DNA brick nanostructures with dendritic oligonucleotides attached to the outer surface. We find that nanostructures assembled from DNA brick motifs remain stable against denaturation without any chemical modifications. Furthermore, densely coating the outer surface of DNA brick nanostructures with dendritic oligonucleotides prevents nuclease digestion.

     
    more » « less
  5. Abstract

    The use of DNA‐based nanomaterials in biomedical applications is continuing to grow, yet more emphasis is being put on the need for guaranteed structural stability of DNA nanostructures in physiological conditions. Various methods have been developed to stabilize DNA origami against low concentrations of divalent cations and the presence of nucleases. However, existing strategies typically require the complete encapsulation of nanostructures, which makes accessing the encased DNA strands difficult, or chemical modification, such as covalent crosslinking of DNA strands. We present a stabilization method involving the synthesis of DNA brick nanostructures with dendritic oligonucleotides attached to the outer surface. We find that nanostructures assembled from DNA brick motifs remain stable against denaturation without any chemical modifications. Furthermore, densely coating the outer surface of DNA brick nanostructures with dendritic oligonucleotides prevents nuclease digestion.

     
    more » « less