Expanding the chemical functionality of DNA nanomaterials generated by rolling circle amplification

Abstract

Rolling circle amplification (RCA) is a powerful tool for the construction of DNA nanomaterials such as hydrogels, high-performance scaffolds and DNA nanoflowers (DNFs), hybrid materials formed of DNA and magnesium pyrophosphate. Such DNA nanomaterials have great potential in therapeutics, imaging, protein immobilisation, and drug delivery, yet limited chemistry is available to expand their functionality. Here, we present orthogonal strategies to produce densely modified RCA products and DNFs. We provide methods to selectively modify the DNA component and/or the protein cargo of these materials, thereby greatly expanding the range of chemical functionalities available to these systems. We have used our methodology to construct DNFs bearing multiple surface aptamers and peptides capable of binding to cancer cells that overexpress the HER2 oncobiomarker, demonstrating their potential for diagnostic and therapeutic applications.

From polymerase engineering to semi-synthetic life: artificial expansion of the central dogma

“…For example, Brown and co-workers employed RCA with base-modified dUTP and dCTP to construct modified DNA nanoflowers, to which various cargos, including fluorophores and functional peptides, could be densely attached, and demonstrated their potential use in diagnostics and therapeutics. 8 UBPs can be employed to increase the number of possible DNA or RNA sequences used for the assembly of nucleic acid nanostructures, and also to make these nanostructures uninvadable to natural DNAs or RNAs. For example, Tan and co-workers recently used DNA sequences containing unnatural bases Z and P to construct an aptamer-nanotrain assembly, and demonstrated its application in drug delivery.…”

“…[2][3][4][5] Numerous polymerase mutants with varied activities of synthesizing, reverse transcribing, and amplifying different unnatural nucleic acids have been obtained and broadly applied in the production and evolution of functional unnatural nucleic acids, including aptamers, catalysts, and nanomaterials. [6][7][8] Other than in vitro application of unnatural nucleic acids, efforts have also been made to introduce unnatural nucleic acid components into living organisms, including creation of semi-synthetic organisms (SSOs) that are able to replicate, transcribe and translate unnatural base pairs (UBPs), which greatly expanded the genetic alphabet in vivo. [9][10][11] Development of unnatural nucleic acids Modifications or alterations have been introduced into different moieties of nucleic acids via chemical synthesis of the structural units, which leads to broad expansion of structures, properties, functions, and applications of nucleic acids.…”

“…of nucleobases modified with functional groups into nucleic acid materials immediately enables the coupling of various molecules onto these materials and thus expands the functionalities of these materials. For example, Brown and coworkers employed RCA with base-modified dUTP and dCTP to construct modified DNA nanoflowers, to which various cargos, including fluorophores and functional peptides, could be densely attached, and demonstrated their potential use in diagnostics and therapeutics8 . UBPs can be employed to increase the number of possible DNA or RNA sequences used for the assembly of nucleic acid nanostructures, and also to make these nanostructures uninvadable to natural DNAs or RNAs.…”

Historical Perspectives and Recent Research on DNA Hydrogel-Based Stimuli-Responsive Systems and Bioengineering Applications

“…Song et al 133 proposed a DNA hydrogel that exhibited RNA-I (interfering gels) and consisted of a plasmid carrying a gene that transcribed siRNA for the target mRNA as part of the gel scaffold. Baker et al 134 offered an orthogonal strategy to produce densely modified RCA products and DNA nanoflowers that attached to cancer cells overexpressing the HER2 tumor biomarkers.…”

DNA‐Dependent Prussian Blue Nanoflowers for Biosensing, Catalysis and Imaging

“…[23,24] The unique structure of DNA showcases its program mability such as systematic variation of charge, size, hydropho bicity, and binding modes. [25][26][27][28][29][30][31] In addition, the affinities of the DNA nucleobases (its nitrogen, oxygen functional groups) to different metal surfaces is crucial for altering metal deposi tion in a sequencedependent manner. [32,33] Hence, DNA has been used as a capping ligand for controlling morphology of nanomaterials in a sequencedependent manner.…”