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

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 (<i>n</i>, <i>m</i>) 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 (<i>n</i> × <i>m</i>) 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

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

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

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.ISSN:1547-6286ISSN:1555-858