Innovative developments and emerging technologies in RNA therapeutics.

RNA-based therapeutics are emerging as a powerful platform for the treatment of multiple diseases. Currently, the two main categories of nucleic acid therapeutics, antisense oligonucleotides and small interfering RNAs (siRNAs), achieve their therapeutic effect through either gene silencing, splicing modulation or microRNA binding, giving rise to versatile options to target pathogenic gene expression patterns. Moreover, ongoing research seeks to expand the scope of RNA-based drugs to include more complex nucleic acid templates, such as messenger RNA, as exemplified by the first approved mRNA-based vaccine in 2020. The increasing number of approved sequences and ongoing clinical trials has attracted considerable interest in the chemical development of oligonucleotides and nucleic acids as drugs, especially since the FDA approval of the first siRNA drug in 2018. As a result, a variety of innovative approaches is emerging, highlighting the potential of RNA as one of the most prominent therapeutic tools in the drug design and development pipeline. This review seeks to provide a comprehensive summary of current efforts in academia and industry aimed at fully realizing the potential of RNA-based therapeutics. Towards this, we introduce established and emerging RNA-based technologies, with a focus on their potential as biosensors and therapeutics. We then describe their mechanisms of action and their application in different disease contexts, along with the strengths and limitations of each strategy. Since the nucleic acid toolbox is rapidly expanding, we also introduce RNA minimal architectures, RNA/protein cleavers and viral RNA as promising modalities for new therapeutics and discuss future directions for the field.

DNA Dendrons as Agents for Intracellular Delivery.

Herein, a method for synthesizing and utilizing DNA dendrons to deliver biomolecules to living cells is reported. Inspired by high-density nucleic acid nanostructures, such as spherical nucleic acids, we hypothesized that small clusters of nucleic acids, in the form of DNA dendrons, could be conjugated to biomolecules and facilitate their cellular uptake. We show that DNA dendrons are internalized by 90% of dendritic cells after just 1 h of treatment, with a >20-fold increase in DNA delivery per cell compared with their linear counterparts. This effect is due to the interaction of the DNA dendrons with scavenger receptor-A on cell surfaces, which results in their rapid endocytosis. Moreover, when conjugated to peptides at a single attachment site, dendrons enhance the cellular delivery and activity of both the model ovalbumin 1 peptide and the therapeutically relevant thymosin alpha 1 peptide. These findings show that high-density, multivalent DNA ligands play a significant role in dictating cellular uptake of biomolecules and consequently will expand the scope of deliverable biomolecules to cells. Indeed, DNA dendrons are poised to become agents for the cellular delivery of many molecular and nanoscale materials.

Defining the Design Parameters for in Vivo Enzyme Delivery Through Protein Spherical Nucleic Acids.

The translation of proteins as effective intracellular drug candidates is limited by the challenge of cellular entry and their vulnerability to degradation. To advance their therapeutic potential, cell-impermeable proteins can be readily transformed into protein spherical nucleic acids (ProSNAs) by densely functionalizing their surfaces with DNA, yielding structures that are efficiently taken up by cells. Because small structural changes in the chemical makeup of a conjugated ligand can affect the bioactivity of the associated protein, structure-activity relationships of the linker bridging the DNA and the protein surface and the DNA sequence itself are investigated on the ProSNA system. In terms of attachment chemistry, DNA-based linkers promote a sevenfold increase in cellular uptake while maintaining enzymatic activity in vitro as opposed to hexaethylene glycol (HEG, Spacer18) linkers. Additionally, the employment of G-quadruplex-forming sequences increases cellular uptake in vitro up to fourfold. When translating to murine models, the ProSNA with a DNA-only shell exhibits increased blood circulation times and higher accumulation in major organs, including lung, kidney, and spleen, regardless of sequence. Importantly, ProSNAs with an all-oligonucleotide shell retain their enzymatic activity in tissue, whereas the native protein loses all function. Taken together, these results highlight the value of structural design in guiding ProSNA biological fate and activity and represent a significant step forward in the development of intracellular protein-based therapeutics.

Automated Synthesis and Purification of Guanidine-Backbone Oligonucleotides.

This protocol describes a method based on iodine and a base as mild coupling reagents to synthetize deoxyribonucleic guanidines (DNGs)-oligodeoxynucleotide analogues with a guanidine backbone. DNGs display unique properties, such as high cellular uptake with low toxicity and increased stability against nuclease degradation, but have been impeded in their development by the requirement for toxic and iterative manual synthesis protocols. The novel synthesis method reported here eliminates the need for the toxic mercuric chloride and pungent thiophenol that were critical to previous DNG synthesis methods and translates their synthesis to a MerMadeTM 12 automated oligonucleotide synthesizer. This method can be used to synthesize DNG strands up to 20 bases in length, along with 5'-DNG-DNA-3' chimeras, at 1- to 5-µmol scales in a fully automated manner. We also present detailed and accessible instructions to adapt the MerMadeTM 12 oligonucleotide synthesizer to enable the parallel synthesis of DNG and DNA/RNA oligonucleotides. Because DNG linkages alter the overall charge of the oligonucleotides, we also describe purification strategies to generate oligonucleotides with varying lengths and numbers of DNGs, based on extraction or preparative-scale gel electrophoresis, along with methods to characterize the final products. Overall, this article provides an overview of the synthesis, purification, and handling of DNGs and mixed-charge DNG-DNA oligonucleotides. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of a MerMadeTM synthesizer for guanidine couplings Basic Protocol 2: Synthesis of DNG strands on a MerMadeTM synthesizer Basic Protocol 3: Purification of DNG strands using preparative acetic acid urea (AU) PAGE Basic Protocol 4: Characterization of DNG strands using MALDI-TOF MS Basic Protocol 5: Characterization of DNG strands using AU PAGE Support Protocol 1: Synthesis of initiator-functionalized CPG Support Protocol 2: Synthesis of thiourea monomer.

Mercury-Free Automated Synthesis of Guanidinium Backbone Oligonucleotides.

A new method for synthesizing deoxynucleic guanidine (DNG) oligonucleotides that uses iodine as a mild and inexpensive coupling reagent is reported. This method eliminates the need for the toxic mercury salts and pungent thiophenol historically used in methods aimed at preparing DNG oligonucleotides. This coupling strategy was readily translated to a standard MerMade 12 oligonucleotide synthesizer with coupling yields of 95% and has enabled the synthesis of a 20-mer DNG oligonucleotide, the longest DNG strand to date, in addition to mixed DNA-DNG sequences with 3-9 DNG inserts. Importantly, DNG oligonucleotides exhibit robust unaided cellular uptake as compared to unmodified oligonucleotides without apparent cellular toxicity. Taken together, these findings should greatly increase the accessibility of cationic backbone modifications and assist in the development of oligonucleotide-based drugs.

Multivalent Cation-Induced Actuation of DNA-Mediated Colloidal Superlattices.

Nanoparticles functionalized with DNA can assemble into ordered superlattices with defined crystal habits through programmable DNA "bonds". Here, we examine the interactions of multivalent cations with these DNA bonds as a chemical approach for actuating colloidal superlattices. Multivalent cations alter DNA structure on the molecular scale, enabling the DNA "bond length" to be reversibly altered between 17 and 3 nm, ultimately leading to changes in the overall dimensions of the micrometer-sized superlattice. The identity, charge, and concentration of the cations each control the extent of actuation, with Ni2+ capable of inducing a remarkable >65% reversible change in crystal volume. In addition, these cations can increase "bond strength", as evidenced by superlattice thermal stability enhancements of >60 °C relative to systems without multivalent cations. Molecular dynamics simulations provide insight into the conformational changes in DNA structure as the bond length approaches 3 nm and show that cations that screen the negative charge on the DNA backbone more effectively cause greater crystal contraction. Taken together, the use of multivalent cations represents a powerful strategy to alter superlattice structure and stability, which can impact diverse applications through dynamic control of material properties, including the optical, magnetic, and mechanical properties.

A Cross-Linking Approach to Stabilizing Stimuli-Responsive Colloidal Crystals Engineered with DNA.

Two DNA-cross-linking reagents, bis-chloroethylnitrosourea and 8-methoxypsoralen, are used to covalently cross-link interstrand base pairs in DNA bonds that, in part, define colloidal crystals engineered with DNA. The irreversible linkages formed increase the chemical and thermal stability of the crystals and do not significantly affect their long-range order, as evidenced by small-angle X-ray scattering data. The post-modified crystals are stable in environments that the pre-modified structures are not, including solvents that encompass a broad range of polarities from ethanol to hexanes, and in aqueous media at pH 0 and 14. Interestingly, the cross-linked DNA bonds within these crystals still retain their flexibility, which is reflected by a solvent-dependent reversible change in lattice parameter. Since these organic cross-linking reagents, in comparison with inorganic approaches (use of silver ions or SiO2), have marginal effects on the composition and properties of the crystals, they provide an attractive alternative for stabilizing colloidal crystals engineered with DNA and make them potentially useful in a broader range of media.

Design Strategy to Access siRNA-Encapsulating DNA "Nanosuitcases" That Can Conditionally Release Their Cargo.

DNA nanotechnology enables the design and assembly of DNA nanostructures with unprecedented control over their size and shape. Additionally, the programmable base-pairing alphabet of DNA allows the incorporation of responsive units within these DNA nanostructures. Here, we describe a general design strategy to construct responsive DNA prisms that can encapsulate and selectively release an encapsulated siRNA upon recognition of an oligonucleotide trigger. This prismatic DNA scaffold design is adaptable and can encapsulate oligonucleotides of any length and type. Moreover, the prism can be made to respond to an oligonucleotide trigger of interest like a messenger RNA (mRNA) or a microRNA (miRNA), thus enabling dual or synergistic therapeutic strategies. We present an overview of the design strategy used to access these DNA nanostructures, followed by the steps involved in DNA sequence generation, assembly, and validation of this construct.

Optimized DNA "Nanosuitcases" for Encapsulation and Conditional Release of siRNA.

We set out to design, synthesize, and optimize a DNA-minimal cage capable of encapsulating oligonucleotide drugs to facilitate their delivery. Through rational design and optimization using in vitro assays, we have assembled the first DNA "nanosuitcase" that can encapsulate a siRNA construct and release it upon recognition of an oligonucleotide trigger. The latter may be a mRNA or a microRNA (miRNA) which offers potential for dual or synergistic therapy. This construct assembles in near 100% yield, releases its cargo on demand, and can sustain biological conditions. Moreover, we find that the DNA scaffold is able to protect its cargo against site-specific cleavage and nuclease degradation. Release of the cargo is performed with fixed cells using a FRET-enabled construct imaged by confocal microscopy and reveals that the DNA cage remains responsive at the molecular level in a complex cellular environment. We foresee this construct will be able to address challenges in drug delivery, more specifically in nontoxic delivery and targeted release.

Controlled growth of DNA structures from repeating units using the vernier mechanism.

In this report, we demonstrate the assembly of length-programmed DNA nanostructures using a single 16 base sequence and its complement as building blocks. To achieve this, we applied the Vernier mechanism to DNA assembly, which uses a mismatch in length between two monomers to dictate the final length of the product. Specifically, this approach relies on the interaction of two DNA strands containing a different number (n, m) of complementary binding sites: these two strands will keep binding to each other until they come into register, thus generating a larger assembly whose length (n × m) is encoded by the number of binding sites in each strand. While the Vernier mechanism has been applied to other areas of supramolecular chemistry, here we present an application of its principles to DNA nanostructures. Using a single 16 base repeat and its complement, and varying the number of repeats on a given DNA strand, we show the consistent construction of duplexes up to 228 base pairs (bp) in length. Employing specific annealing protocols, strand capping, and intercalator chaperones allows us to further grow the duplex to 392 base pairs. We demonstrate that the Vernier method is not only strand-efficient, but also produces a cleaner, higher-yielding product than conventional designs.

Intercalators as molecular chaperones in DNA self-assembly.

DNA intercalation has found many diagnostic and therapeutic applications. Here, we propose the use of simple DNA intercalators, such as ethidium bromide, as tools to facilitate the error-free self-assembly of DNA nanostructures. We show that ethidium bromide can influence DNA self-assembly, decrease the formation of oligomeric side products, and cause libraries of multiple equilibrating structures to converge into a single product. Using a variety of 2D- and 3D-DNA systems, we demonstrate that intercalators present a powerful alternative for the adjustment of strand-end alignment, favor the formation of fully duplexed "closed" structures, and create an environment where the smallest, most stable structure is formed. A new 3D-DNA motif, the ninja star, was self-assembled in quantitative yield with this method. Moreover, ethidium bromide can be readily removed using isoamyl alcohol extractions combined with intercalator-specific spin columns, thereby yielding the desired ready-to-use DNA structure.

Rolling circle amplification-templated DNA nanotubes show increased stability and cell penetration ability.

DNA nanotubes hold promise as scaffolds for protein organization, as templates of nanowires and photonic systems, and as drug delivery vehicles. We present a new DNA-economic strategy for the construction of DNA nanotubes with a backbone produced by rolling circle amplification (RCA), which results in increased stability and templated length. These nanotubes are more resistant to nuclease degradation, capable of entering human cervical cancer (HeLa) cells with significantly increased uptake over double-stranded DNA, and are amenable to encapsulation and release behavior. As such, they represent a potentially unique platform for the development of cell probes, drug delivery, and imaging tools.