The Emergence of Oligonucleotide Building Blocks in the Multispecific Proximity-Inducing Drug Toolbox of Destruction.

Oligonucleotides are a rapidly emerging class of therapeutics. Their most well-known examples are informational drugs that modify gene expression by binding mRNA. Despite inducing proximity between biological machinery and mRNA when applied to modulating gene expression, oligonucleotides are not typically labeled as "proximity-inducing" in literature.

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.

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 MerMade 12 automated oligonucleotide synthesizer.

Defining the Design Parameters for 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.

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.

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.

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.

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.

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.

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.

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.

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.