The Use of Minimal RNA Toeholds to Trigger the Activation of Multiple Functionalities

Afonin, Kirill A.; Viard, Matthias; Tedbury, Philip R.; Bindewald, Eckart; Parlea, Lorena; Howington, Marshall; Valdman, Melissa; Johns-Boehme, Alizah; Brainerd, Cara; Freed, Eric O.; Shapiro, Bruce A.

doi:10.1021/acs.nanolett.5b04676

Cited by 40 publications

(27 citation statements)

References 48 publications

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“…The intra-tile crossover distance is two full RNA helical turns (22 bp) and the inter-tile crossover distance is about two and a half helical turns (28 bp), as described in previous work 30 . To functionalize the tile, Dicer Substrate (DS) RNAs 31 , that provide Dicer-assisted intracellular release of the siRNAs 32,33 , were introduced. For that, tile strands (Sc) were extended with antisense strands of DS RNAs.…”

Section: Resultsmentioning

confidence: 99%

Programmable RNA microstructures for coordinated delivery of siRNAs

et al. 2016

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RNA is a natural multifunctional polymer, and is an essential component in both complex pathways and structures within the cellular environment. For this reason, artificial self-assembling RNA nanostructures are emerging as a powerful tool with broad applications in drug delivery and metabolic pathway regulation. To date, coordinated delivery of functional molecules via programmable RNA assemblies has been primarily done using nanosize RNA scaffolds. However, larger scaffolds could expand existing capabilities for spatial arrangement of ligands, and enable the controlled delivery of highly concentrated molecular loads. Here, we investigate whether micron-size RNA scaffolds can be assembled and further functionalized with different cargos (e.g. various siRNAs and fluorescent tags) for their synchronized delivery to diseased cells. Since known design approaches to build large RNA scaffolds are still underdeveloped, we apply, for the first time, a tiling method widely used in DNA nanotechnology. DNA tiles have been extensively used to build a variety of scalable and modular structures that are easily decorated with other ligands. Here, we adapt a double crossover (DX) DNA tile motif to design de novo DX RNA tiles that assemble and form lattices via programmed sticky end interactions. Because the assembly protocols used in DNA nanotechnology are not suitable for RNA assembly, we optimize them to guarantee high yield of RNA lattices. The resulting constructs are robust and modular with respect to the presence of distinct siRNAs and fluorophores. RNA tiles and lattices are successfully transfected in either human breast cancer or prostate cancer cells, where they efficiently knockdown the expression of target genes. Blood serum stability assays indicate that RNA lattices are more resilient to nuclease degradation when compared to individual tiles, thus making them better suited for therapeutic purposes. Overall, because of its design simplicity, we anticipate that this approach will be utilized for a wide range of applications in therapeutic RNA nanotechnology.

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Section: Resultsmentioning

confidence: 99%

Programmable RNA microstructures for coordinated delivery of siRNAs

et al. 2016

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“…RNA/DNA hybrid nanoparticles have been utilized as multifunctional drug delivery carriers 19–21 . The phi29 pRNA three-way junction (3WJ) motif with unusually robust thermostable properties 22,23 is used as an platform to construct a new generation of therapeutic nanoparticles 23–25 .…”

Section: Introductionmentioning

confidence: 99%

RNA nanoparticles harboring annexin A2 aptamer can target ovarian cancer for tumor-specific doxorubicin delivery

Zhang

et al. 2017

Nanomedicine: Nanotechnology, Biology and Medicine

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A novel modified nucleic acid nanoparticle harboring an annexin A2 aptamer for ovarian cancer cell targeting and a GC rich sequence for doxorubicin loading is designed and constructed. The system utilizes a highly stable three-way junction (3WJ) motif from phi29 packaging RNA as a core structure. A phosphorothioate-modified DNA aptamer targeting annexin A2, Endo28, was conjugated to one arm of the 3WJ. The pRNA-3WJ motif retains correct folding of attached aptamer, keeping its functions intact. It is of significant utility for aptamer-mediated targeted delivery. The DNA/RNA hybrid nanoparticles remained intact after systemic injection in mice and strongly bound to tumors with little accumulation in healthy organs 6 hours post-injection. The Endo28-3WJ-Sph1/Dox intercalates selectively enhanced toxicity to annexin A2 positive ovarian cancer cells in vitro. The constructed RNA/DNA hybrid nanoparticles can potentially enhance the therapeutic efficiency of doxorubicin at low doses for ovarian cancer treatment through annexin A2 targeted drug delivery.

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“…RNA structures are diverse and can prohibit or promote interactions with other RNAs, proteins, and metabolites to enable a broad range of RNA function. For example, bacterial RNA structures inhibit transcription elongation (Jagadeeswaran et al, 2010), translation initiation (Afonin et al, 2016), and RNA degradation (Hui et al, 2015).. In eukaryotes, there is growing evidence that RNA structure impacts gene expression processes (Rouskin et al, 2014;Ding et al, 2014;Spitale et al, 2015;Talkish et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

DUETT quantitatively identifies known and novel events in nascent RNA structural dynamics from chemical probing data

Xue

Lucks

et al. 2018

Preprint

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Motivation: RNA molecules can undergo complex structural dynamics, especially during transcription, which influence their biological functions. Recently developed high-throughput chemical probing experiments study RNA cotranscriptional folding to generate nucleotide-resolution 'reactivities' for each length of a growing nascent RNA and reflect structural dynamics. However, the manual annotation and qualitative interpretation of reactivity across these large datasets can be nuanced, laborious, and difficult for new practitioners. We developed a quantitative and systematic approach to automatically detect RNA folding events from these datasets to reduce human bias/error, standardize event discovery, and generate hypotheses about RNA folding trajectories for further analysis and experimental validation. Results: Detection of Unknown Events with Tunable Thresholds (DUETT) identifies RNA structural transitions in cotranscriptional RNA chemical probing datasets. DUETT employs a feedback control-inspired method and a linear regression approach and relies on interpretable and independently tunable parameter thresholds to match qualitative user expectations with quantitatively identified folding events. We validate the approach by identifying known RNA structural transitions within the cotranscriptional folding pathways of the Escherichia coli signal recognition particle (SRP) RNA and the Bacillus cereus crcB fluoride riboswitch. We identify previously overlooked features of these datasets such as heightened reactivity patterns in the SRP RNA about 12 nucleotide lengths before base pair rearrangement. We then apply a sensitivity analysis to identify tradeoffs when choosing parameter thresholds. Finally, we show that DUETT is tunable across a wide range of contexts, enabling flexible application to study broad classes of RNA folding mechanisms.This supplementary file contains the methods for DUETT, results from the automated threshold selection process, results from averaging replicates then conducting event detection, additional sensitivity analysis, a table of assumptions, and a table of userdefined threshold parameters used in the SHAPE-Seq event detector manuscript. This word document also contains descriptions for Supplementary Files 1-3.

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The Use of Minimal RNA Toeholds to Trigger the Activation of Multiple Functionalities

Cited by 40 publications

References 48 publications

Programmable RNA microstructures for coordinated delivery of siRNAs

Programmable RNA microstructures for coordinated delivery of siRNAs

RNA nanoparticles harboring annexin A2 aptamer can target ovarian cancer for tumor-specific doxorubicin delivery

DUETT quantitatively identifies known and novel events in nascent RNA structural dynamics from chemical probing data

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