Nucleic acid nanostructures for in vivo applications: The influence of morphology on biological fate

Abstract

The development of programmable biomaterials for use in nanofabrication represents a major advance for the future of biomedicine and diagnostics. Recent advances in structural nanotechnology using nucleic acids have resulted in dramatic progress in our understanding of nucleic acid-based nanostructures (NANs) for use in biological applications. As the NANs become more architecturally and functionally diverse to accommodate introduction into living systems, there is a need to understand how critical design features can be controlled to impart desired performance in vivo. In this review, we survey the range of nucleic acid materials utilized as structural building blocks (DNA, RNA, and xenonucleic acids), the diversity of geometries for nanofabrication, and the strategies to functionalize these complexes. We include an assessment of the available and emerging characterization tools used to evaluate the physical, mechanical, physiochemical, and biological properties of NANs in vitro. Finally, the current understanding of the obstacles encountered along the in vivo journey is contextualized to demonstrate how morphological features of NANs influence their biological fates. We envision that this summary will aid researchers in the designing novel NAN morphologies, guide characterization efforts, and design of experiments and spark interdisciplinary collaborations to fuel advancements in programmable platforms for biological applications.

FIG. 1.

An overview of key parameters to consider for the design of nucleic acid nanostructures for biological applications. Critical features that can be tuned include (a) the choice of NA material, (b) morphological features including size and shape, (c) physiochemical properties of the structure, and (d) any modifications to the surface to facilitate delivery or include ligands for functional applications. These features directly impact (e) the in vivo fate of the structures. Part (d) is modified with permission from Jiang et al., Chem 7(5), 1156–1179 (2021). Copyright 2021 Elsevier. Part (e) is created using BioRender.

FIG. 2.

An overview of different DNA design strategies. (a) DNA tiles are designed with sticky ends to self-assemble into a network structure. (b) Framework DNA objects are assembled using the addition of several component oligonucleotides. (c) The DNA origami method involves the use of a uniform scaffold, that is, folded through addition of partly complementary staple strands. (d) Wireframe DNA origami is a method that combines the DNA origami annealing method with the design of framework objects to form more stable wireframe architectures. Modified with permission from Jiang et al., Chem 7(5), 1156–1179. (2021). Copyright 2021 Elsevier.

FIG. 3.

An overview of common RNA design motifs. (a) Commonly utilized fundamental motifs in RNA nanofabrication include the kink tun, kissing loop, three-way loop, three-way junction, and four-way junction. (b) Similar to DNA, framework RNA objects are assembled from the addition of several complementary oligonucleotides. (c) RNA origami structures can be fabricated through methods similar to DNA origami with crossover (CX) regions or they can also be assembled by a single strand and feature loop (L) and kissing loop (KL) regions. Part (a) modified with permission from Li et al., Nature 9, 2196 (2018). Copyright 2018 Springer Nature, licensed under a Creative Commons Attribution (CC-BY) license.

FIG. 4.

Examples of xenonucleic acids (XNAs) used in nanotechnology applications. (a) Molecular structures of XNAs with modifications to either the sugars (orange), phosphates (blue), or total backbone replacement (green). (b) Crystal structures of naturally occurring NA duplexes in comparison to the geometry of homoduplexes and heteroduplexes of XNAs with DNA or RNA. Part (b) is reproduced with permission from Anosova and Kowal, Nucleic Acids Res. 44(3), 1007–1021 (2015). Copyright 2015 Oxford Press.

FIG. 5.

An overview of the workflow for the design and validation process to develop new NANs for in vivo applications. Researchers first begin with a set of design requirements and constraints that match the needs of their targeted applications. Structures are designed, synthesized, and assembled, with characterization techniques used for validation at each step along the process. Results are reviewed intermittently to determine agreement with predetermined design requirements. The in vitro cycle continues until the requirements are satisfied, then in vivo evaluation is performed. This process is not always straightforward, and often many iterations of design and assembly are needed before validation experiments can be initiated. This figure was created using BioRender.

FIG. 6.

Overview of the morphological features of NANs used in biological applications. NANs can be assembled in 2D (a) or 3D (b) morphologies depending on the desired goals of stability and size. (c) Structural features linked with improved stability include increasing helix layers, increasing the number of strand crossovers, restricting topology to minimize single-stranded overhangs, and using NA analogs with higher melting temperatures. (d) An example of the assembly of a complex dynamic NAN in the form of a shape-changing DNA origami tubular nanorobot for delivery of a thrombin cargo following nucleolin binding in tumor microvasculature. Part (a) reproduced with permissions from Khisamutdinov et al., Nucleic Acids Res. 42, 15 (2014). Copyright 2014 Oxford University Press, licensed under a Creative Commons Attribution (CC-BY) license and Jiang et al., Nat. Biomed. Eng. 2, 865–877 (2018). Copyright 2018 Springer Nature. Part (b) reproduced with permissions from Li et al., Adv. Mater. 28, 34 (2016). Copyright 2016 John Wiley and Sons; Høiberg et al., Biotechnol. J. 14, 1700634 (2018). Copyright 2018 John Wiley and Sons; Bastings et al., Nano Lett. 18(6), 3557–3564 (2018). Copyright 2018 American Chemical Society; and Ijäs et al., ACS Nano 13(5), 5959–5967 (2019). Copyright 2019 American Chemical Society, licensed under a Creative Commons Attribution (CC-BY) license. Part (d) is modified with permission from Li et al., Nat. Biotechnol. 36, 258–264 (2018). Copyright 2018 Springer Nature.

FIG. 7.

Examples of functionalization strategies to improve biological properties of NANs. Modifications to the NAN architecture can be utilized to (a) enable specific functions, such as imaging or stimulation of the immune system, (b) protect the structure from degradation, and (c) facilitate transport to the intended biological location. Part (a) reproduced with permissions by Jiang et al., ACS Appl. Mater. Interfaces 8(7), 4378–4384 (2016). Copyright 2016 American Chemical Society; Veneziano et al., Nat. Nanotechnol. 15, 716–723 (2020). Copyright 2020 Springer Nature; and Li et al., ACS Nano 5(11), 8783–8789 (2011). Copyright 2011 American Chemical Society. Part (b) reproduced with permission by Kiviaho et al., Nanoscale 8, 11674–11680 (2016). Copyright 2016 Royal Society of Chemistry, licensed under a Creative Commons Attribution (CC-BY-NC) license; Kim and Yin, Angew. Chem., Int. Ed., 59, 700–703 (2019). Copyright 2019 John Wiley and Sons; and Perrault and Shih, ACS Nano 8(5), 5132–5140 (2014). Copyright 2014 American Chemical Society. Part (c) reproduced with permission from Whitehouse et al., Bioconjugate Chem. 30(7), 1836–1844 (2019). Copyright 2019 American Chemical Society; Sakai et al., Genes 9, 571 (2018). Copyright 2018 MDPI, licensed under a Creative Commons Attribution (CC-BY) license; and Xia et al., Biochemistry 55(9), 1326–1331 (2016). Copyright 2016 American Chemical Society.

FIG. 8.

Two primary challenges faced by NANs in the bloodstream are nuclease degradation and formation of a protein corona. (a) NANs are susceptible to cleavage by endogenous extracellular nucleases in blood and other biofluids. (b) Proteins within the blood interact with and adsorb onto the surface of NANs based on hydrophobicity and charge properties in patterns that are often challenging to predict.

FIG. 9.

(a) An overview of mechanisms of cell internalization utilized by NANs. (b) The efficiency and extent of uptake can be enhanced due to modifications to the NAN morphology, such as increasing the compactness, size, and 3D-character. Functional modifications, including cholesterol or other hydrophobic moieties, cationic proteins, aptamers, or cell penetrating peptides, can further increase uptake. Part (b) is modified with permissions by Bastings et al., Nano Lett. 18(6), 3557–3564 (2018). Copyright 2018 American Chemical Society; Wang et al., J. Am. Chem. Soc. 140(7), 2478–2484 (2018), Copyright 2018 American Chemical Society; and Zeng et al., J. Mater. Chem. B 6. 1605–1612 (2018). Copyright 2018 Royal Society of Chemistry.

FIG. 10.

(a) The intracellular fates of NANs can follow one of several pathways, with the primary route following caveolin-mediated endocytosis as the endo-lysosomal route to degradation. Alternative routes (in gray) include functional trafficking and endosomal escape to the cytoplasm, which are modulated by the NAN morphology and functionalization. (b) Immunomodulation in the endosome occurs through toll-like receptors specific to DNA and RNA, leading to the production of proinflammatory signals (e.g., cytokines).

FIG. 11.

Examples of the biodistribution of NANs in mouse models. (a) PET images of the biodistribution of three ⁶⁴Cu-labeled objects in healthy mice. The M13 ssDNA scaffold shows RES-mediated removal and accumulation in the liver, while the flexible DNA origami rectangle (Rec-DON) and triangle (Tri-DON) show preferential accumulation in the kidneys. Reproduced with permission from Jiang et al., Nat. Biomed. Eng. 2, 865–877 (2018). Copyright 2018 Springer Nature. (b) Comparison of the biodistribution of sequence-identical tetrahedral NANs made of DNA (D-sTd), L-DNA (L-sTd), 2′-O-Me-RNA (M-sTd), and 2′-F-RNA (F-sTd). The L-DNA structure revealed the longest circulation lifetime of all four structures with preferential kidney accumulation. Reproduced with permission from Thai et al., ACS Central Sci. 6(12), 2250–2258 (2020). Copyright 202 American Chemical Society.