Subha R. Das

Click Chemistry for Rapid Labeling and Ligation of RNA

The copper(I)‐promoted azide–alkyne cycloaddition reaction (click chemistry) is shown to be compatible with RNA (with free 2′‐hydroxyl groups) in spite of the intrinsic lability of RNA. RNA degradation is minimized through stabilization of the CuI in aqueous buffer with acetonitrile as cosolvent and no other ligand; this suggests the general possibility of “ligandless” click chemistry. With the viability of click chemistry validated on synthetic RNA bearing “click”‐reactive alkynes, the scope of the reaction is extended to in‐vitro‐transcribed or, indeed, any RNA, as a click‐reactive azide is incorporated enzymatically. Once clickable groups are installed on RNA, they can be rapidly click labeled or conjugated together in click ligations, which may be either templated or nontemplated. In click ligations the resultant unnatural triazole‐linked RNA backbone is not detrimental to RNA function, thus suggesting a broad applicability of click chemistry in RNA biological studies.

RNA labeling, conjugation and ligation.

Advances in RNA nanotechnology will depend on the ability to manipulate, probe the structure and engineer the function of RNA with high precision. This article reviews current abilities to incorporate site-specific labels or to conjugate other useful molecules to RNA either directly or indirectly through post-synthetic labeling methodologies that have enabled a broader understanding of RNA structure and function. Readily applicable modifications to RNA can range from isotopic labels and fluorescent or other molecular probes to protein, lipid, glycoside or nucleic acid conjugates that can be introduced using combinations of synthetic chemistry, enzymatic incorporation and various conjugation chemistries. These labels, conjugations and ligations to RNA are quintessential for further investigation and applications of RNA as they enable the visualization, structural elucidation, localization, and biodistribution of modified RNA.

Preparation of cationic nanogels for nucleic acid delivery.

Cationic nanogels with site-selected functionality were designed for the delivery of nucleic acid payloads targeting numerous therapeutic applications. Functional cationic nanogels containing quaternized 2-(dimethylamino)ethyl methacrylate and a cross-linker with reducible disulfide moieties (qNG) were prepared by activators generated by electron transfer (AGET) atom transfer radical polymerization (ATRP) in an inverse miniemulsion. Polyplex formation between the qNG and nucleic acid exemplified by plasmid DNA (pDNA) and short interfering RNA (siRNA duplexes) were evaluated. The delivery of polyplexes was optimized for the delivery of pDNA and siRNA to the Drosophila Schneider 2 (S2) cell-line. The qNG/nucleic acid (i.e., siRNA and pDNA) polyplexes were found to be highly effective in their capabilities to deliver their respective payloads.

Star polymers with a cationic core prepared by ATRP for cellular nucleic acids delivery.

Poly(ethylene glycol) (PEG)-based star polymers with a cationic core were prepared by atom transfer radical polymerization (ATRP) for in vitro nucleic acid (NA) delivery. The star polymers were synthesized by ATRP of 2-(dimethylamino)ethyl methacrylate (DMAEMA) and ethylene glycol dimethacrylate (EGDMA). Star polymers were characterized by gel permeation chromatography, zeta potential, and dynamic light scattering. These star polymers were combined with either plasmid DNA (pDNA) or short interfering RNA (siRNA) duplexes to form polyplexes for intracellular delivery. These polyplexes with either siRNA or pDNA were highly effective in NA delivery, particularly at relatively low star polymer weight or molar ratios, highlighting the importance of NA release in efficient delivery systems.

Well-defined biohybrids using reversible-deactivation radical polymerization procedures

The use of reversible deactivation radical polymerization (RDRP) methods has significantly expanded the field of bioconjugate synthesis. RDRP procedures have allowed the preparation of a broad range of functional materials that could not be realized using prior art poly(ethylene glycol) functionalization. The review of procedures for synthesis of biomaterials is presented with a special focus on the use of RDRP to prepare biohybrids with proteins, DNA and RNA.

Solid-Phase Incorporation of an ATRP Initiator for Polymer–DNA Biohybrids†

The combination of polymers with nucleic acids leads to materials with significantly advanced properties. To obviate the necessity and complexity of conjugating two macromolecules, a polymer initiator is described that can be directly covalently linked to DNA during solid-phase synthesis. Polymer can then be grown from the DNA bound initiator, both in solution after the DNA-initiator is released from the solid support as well as directly on the solid support, simplifying purification. The resulting polymer-DNA hybrids were examined by chromatography and fluorescence methods that attested to the integrity of hybrids and the DNA. The ability to use DNA-based supports expands the range of readily available molecules that can be used with the initiator, as exemplified by direct synthesis of a biotin polymer hybrid on solid-support. This method expands the accessibility and range of advanced polymer biohybrid materials.

A protein-polymer hybrid mediated by DNA.

Protein-polymer hybrids (PPHs) represent an important and rapidly expanding class of biomaterials. Typically in these hybrids the linkage between the protein and the polymer is covalent. Here we describe a straightforward approach to a noncovalent PPH that is mediated by DNA. Although noncovalent, the DNA-mediated approach affords the highly specific pairing and assembly properties of DNA. To obtain the protein-DNA conjugate for assembly of the PPH, we report here the first direct copper catalyzed azide-alkyne cycloaddition-based protein-DNA conjugation. This significantly simplifies access to protein-DNA conjugates. The protein-DNA conjugate and partner polymer-DNA conjugate are readily assembled through annealing of the cDNA strands to obtain the PPH, the assembly of which was confirmed via dynamic light scattering and fluorescence spectroscopy.

Autotransfecting Short Interfering RNA through Facile Covalent Polymer Escorts

Short interfering ribonucleic acids (siRNAs) are important agents for RNA interference (RNAi) that have proven useful in gene function studies and therapeutic applications. However, the efficacy of exogenous siRNAs for gene knockdown remains hampered by their susceptibility to cellular nucleases and impermeability to cell membranes. We report here new covalent polymer-escort siRNA constructs that address both of these constraints simultaneously. By simple postsynthetic click conjugation of polymers to the passenger strand of an siRNA duplex followed by annealing with the complementary guide strand, we obtained siRNA in which one strand includes terminal polymer escorts. The polymer escorts both confer protection against nucleases and facilitate cellular internalization of the siRNA. These autotransfecting polymer-escort siRNAs are viable in RNAi and effective in knocking down reporter and endogenous genes.

Automated Synthesis of Well-Defined Polymers and Biohybrids by Atom Transfer Radical Polymerization Using a DNA Synthesizer

A DNA synthesizer was successfully employed for preparation of well-defined polymers by atom transfer radical polymerization (ATRP), in a technique termed AutoATRP. This method provides well-defined homopolymers, diblock copolymers, and biohybrids under automated photomediated ATRP conditions. PhotoATRP was selected over other ATRP methods because of mild reaction conditions, ambient temperature, tolerance to oxygen, and no need to introduce reducing agents or radical initiators. Both acrylate and methacrylate monomers were successfully polymerized with excellent control in the DNA synthesizer. Diblock copolymers were synthesized with different targeted degrees of polymerization and with high retention of chain-end functionality. Both hydrophobic and hydrophilic monomers were grafted from DNA. The DNA-polymer hybrids were characterized by SEC and DLS. The AutoATRP method provides an efficient route to prepare a range of different polymeric materials, especially polymer-biohybrids.

Bright Fluorescent Nanotags from Bottlebrush Polymers with DNA-Tipped Bristles.

Bright signal outputs are needed for fluorescence detection of biomolecules at their native expression levels. Increasing the number of labels on a probe often results in crowding-induced self-quenching of chromophores, and maintaining the function of the targeting moiety (e.g., an antibody) is a concern. Here we demonstrate a simple method to accommodate thousands of fluorescent dye molecules on a single antibody probe while avoiding the negative effects of self-quenching. We use a bottlebrush polymer from which extend hundreds of duplex DNA strands that can accommodate hundreds of covalently attached and/or thousands of noncovalently intercalated fluorescent dyes. This polymer–DNA assembly sequesters the intercalated fluorophores against dissociation and can be tethered through DNA hybridization to an IgG antibody. The resulting fluorescent nanotag can detect protein targets in flow cytometry, confocal fluorescence microscopy, and dot blots with an exceptionally bright signal that compares favorably to commercially available antibodies labeled with organic dyes or quantum dots.