Eduardo Paredes

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.

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.

Backbone-branched DNA building blocks for facile angular control in nanostructures

Nanotechnology based on the highly specific pairing of nucleobases in DNA has been used to generate a wide variety of well-defined two- and three-dimensional assemblies, both static and dynamic. However, control over the junction angles to achieve them has been limited. To achieve higher order assemblies, the strands of the DNA duplex are typically made to deviate at junctions with configurations based on crossovers or non-DNA moieties. Such strand crossovers tend to be intrinsically unstructured with the overall structural rigidity determined by the architecture of the nanoassembly, rather than the junction itself. Specific approaches to define nanoassembly junction angles are based either on the cooperative twist- and strain-promoted tuning of DNA persistence length leading to bent DNA rods for fairly large nano-objects, or de novo synthesis of individual junction inserts that are typically non-DNA and based on small organic molecules or metal-coordinating ligand moieties. Here, we describe a general strategy for direct control of junction angles in DNA nanostructures that are completely tunable about the DNA helix. This approach is used to define angular vertices through readily accessible backbone-branched DNAs (bbDNAs). We demonstrate how such bbDNAs can be used as a new building block in DNA nanoconstruction to obtain well-defined nanostructures. Angular control through readily accessible bbDNA building block provides a general and versatile approach for incorporating well-defined junctions in nanoconstructs and expands the toolkit toward achieving strain free, highly size- and shape-tunable DNA based architectures.

Crystal structure of the Entamoeba histolytica RNA lariat debranching enzyme EhDbr1 reveals a catalytic Zn2+/Mn2+ heterobinucleation

The RNA lariat debranching enzyme, Dbr1, is a metallophosphoesterase that cleaves 2′‐5′ phosphodiester bonds within intronic lariats. Previous reports have indicated that Dbr1 enzymatic activity is supported by diverse metal ions including Ni2+, Mn2+, Mg2+, Fe2+, and Zn2+. While in initial structures of the Entamoeba histolytica Dbr1 only one of the two catalytic metal‐binding sites were observed to be occupied (with a Mn2+ ion), recent structures determined a Zn2+/Fe2+ heterobinucleation. We solved a high‐resolution X‐ray crystal structure (1.8 Å) of the E. histolytica Dbr1 and determined a Zn2+/Mn2+ occupancy. ICP‐AES corroborate this finding, and in vitro debranching assays with fluorescently labeled branched substrates confirm activity.

The Diverse Active Sites in Splicing, Debranching, and MicroRNA Processing Around RNA Phosphodiester Bonds

The cleavage and ligation reactions at RNA phosphodiester bonds are the central reactions catalyzed by enzymes in critical cellular regulatory pathways. In pre-mRNA splicing, two phospho-transesterifications result in the right mRNA for protein synthesis with the intervening intron removed as a lariat structure. The lariat RNA is then debranched by an enzyme that specifically acts on this 2′-5′-branched RNA. Following debranching, some of these introns that include pre-microRNA sequences can be processed by Dicer that cleaves the RNA to provide microRNAs. Dicer and Drosha, enzymes that act on much bigger primary transcripts, are both RNase III-like enzymes that cleave the RNA phosphodiester linkage. All these reactions are in related pathways, and the RNA phosphodiester bonds are most likely cleaved with the aid of two metal ions, yet the active sites that host these could be composed entirely of RNA or entirely of protein, or possibly a hybrid of the two. Where unknown, it is possible to estimate some of these active site architectures through homology to closely related enzymes. Better insight into these related process and active sites will play a key role in leveraging these important RNA regulatory processes for molecular medicine.