Synthesis of folate-labeled siRNAs from a folate derivative phosphoramidite.

With the recent success of GalNAc and the need for extra-hepatic RNAi delivery systems, other receptor-targeting ligands, like folate, have gained increased attention. The folate receptor is an important molecular target in cancer research, as it is overexpressed on numerous tumours while having limited expression in non-malignant tissues. Despite the promise of folate conjugation as a delivery platform in cancer therapeutics, its application in RNAi has been limited by sophisticated, and often expensive, chemistry. Here, we report a straightforward and cost-effective strategy to synthesize a novel folate derivative phosphoramidite for siRNA incorporation. In the absence of a transfection carrier, these siRNAs were selectively taken up by folate receptor-expressing cancer cell lines and displayed potent gene-silencing activity.

SiRNAs with Neutral Phosphate Triester Hydrocarbon Tails Exhibit Carrier-Free Gene-Silencing Activity.

Short interfering RNAs (siRNAs) show promise as gene-silencing therapeutics, but their cellular uptake remains a challenge. We have recently shown the synthesis of siRNAs bearing a single neutral phenylethyl phosphotriester linkage within the sense strand. Here, we report the synthesis of siRNAs bearing three different hydrophobic phosphate triester linkages at key positions within the sense strand and assess their gene silencing in the absence of a transfection carrier. The best siRNAs bearing hydrophobic phosphate triester tails were not aromatic and exhibited effective gene silencing (IC50 ≈ 56-141 nM), whereas the aromatic derivative with three hydrophobic tails did not exhibit carrier-free gene silencing.

Building siRNAs with Cubes: Synthesis and Evaluation of Cubane-Modified siRNAs.

Cubane molecules hold great potential for medicinal chemistry applications due to their inherent stability and low toxicity. In this study, we report the synthesis of a cubane derivative phosphoramidite for the incorporation of cubane into small interfering RNAs (siRNAs). Synthetic siRNAs rely on chemical modifications to improve their pharmacokinetic profiles. However, they are still able to mediate sequence-specific gene silencing via the endogenous RNA interference pathway. We designed a library of siRNAs bearing cubane at different positions within the sense and antisense strands. All siRNAs showed excellent gene-silencing activity, with IC50 values ranging from 45.4 to 305 pM. Incorporating the cubane modification in both the sense and antisense strand led to viable duplexes with good biological activity. To the best of our knowledge, this is the first report of siRNAs bearing a cubane derivative within the backbone.

Static vs. dynamic methods of delivery for science communication: A critical analysis of user engagement with science on social media.

Science communication has been increasingly viewed as a necessity and obligation of scientists in recent years. The rise of Web 2.0 technologies, such as social media, has made communication of science to the public more accessible as a whole. While one of the primary goals of science communication is to increase public engagement, there is very little research to show the type of communication that fosters the highest levels of engagement. Here we evaluate two social medial platforms, Instagram and TikTok, and assess the type of educational science content (ESC) that promotes user awareness and overall engagement. Specifically, we measured the level of engagement between static and dynamic posts on Instagram, and lecture-style and experimental videos on TikTok. User engagement is measured through the analysis of relative number of likes, comments, shares, saves, and views of each post in the various categories. We found that users interact with ESC significantly more (p<0.05) when the content is presented in dynamic ways with a component of experimentation. Together, we took the findings of this study and provided a series of suggestions for conducting science communication on social media, and the type of ESC that should be used to promote better user outcomes.

Synthesis and evaluation of modified siRNA molecules containing a novel glucose derivative.

Chemical modifications are critical for the development of safe and effective siRNAs for downstream applications. In this study, we report the synthesis of a novel glucose phosphoramidite, a triazole-linked to uracil at position one, for incorporation into oligonucleotides. Biological testing revealed that the glucose derivative at key positions within the sense or antisense strand can lead to potent gene-silencing activity, thus highlighting its tolerance in both sense and antisense positions. Furthermore, the A-form helical formation was maintained with this modification. Overall, placing the modification at the 3' end and at key internal positions led to effective RNAi gene-silencing activity.

To Conjugate or to Package? A Look at Targeted siRNA Delivery Through Folate Receptors.

RNA interference (RNAi) applications have evolved from experimental tools to study gene function to the development of a novel class of gene-silencing therapeutics. Despite decades of research, it was not until August 2018 that the US FDA approved the first-ever RNAi drug, marking a new era for RNAi therapeutics. Although there are many limitations associated with the inherent structure of RNA, delivery to target cells and tissues remains the most challenging. RNAs are unable to diffuse across cellular membranes due to their large size and polyanionic backbone and, therefore, require a delivery vector. RNAi molecules can be conjugated to a targeting ligand or packaged into a delivery vehicle. Alnylam has used both strategies in their FDA-approved formulations to achieve efficient delivery to the liver. To harness the full potential of RNAi therapeutics, however, we must be able to target additional cells and tissues. One promising target is the folate receptor α, which is overexpressed in a variety of tumors despite having limited expression and distribution in normal tissues. Folate can be conjugated directly to the RNAi molecule or used to functionalize delivery vehicles. In this review, we compare both delivery strategies and discuss the current state of research in the area of folate-mediated delivery of RNAi molecules.

Effects of Chemical Modifications on siRNA Strand Selection in Mammalian Cells.

Small interfering RNAs (siRNAs) enable efficient gene silencing through RNA interference (RNAi) mechanisms. The RNAi machinery relies on an RNA-guided nuclease, Argonaute-2 (Ago2), which preferentially selects a single strand from an siRNA duplex. Complementarity between the selected strand and an RNA target strand leads to silencing through cleavage. The U.S. Food and Drug Administration's recent approval of two siRNA drugs has reignited optimism for RNAi therapeutics. Despite this recent success in the field, off-target effects are still a major concern; however, chemical modifications have shown promise in mitigating some off-target gene silencing. To evaluate the impact of novel chemical modifications on strand selection, we developed a quantitative polymerase chain reaction-based assay that is compatible with several pre-existing siRNA libraries and was used to characterize chemically modified siRNAs. siRNAs bearing azobenzene and propargyl modifications at the central region of the passenger strand significantly improved strand selection. On the other hand, folic acid-modified siRNAs improved strand selection best when placed at the 3' terminus. This study highlights the development and utility of a convenient method to evaluate the impact that novel chemical modifications have on strand-specific gene silencing of siRNAs.

Targeted delivery and enhanced gene-silencing activity of centrally modified folic acid-siRNA conjugates.

One of the major hurdles in RNAi research has been the development of safe and effective delivery systems for siRNAs. Although various chemical modifications have been proposed to improve their pharmacokinetic behaviour, their delivery to target cells and tissues presents many challenges. In this work, we implemented a receptor-targeting strategy to selectively deliver siRNAs to cancer cells using folic acid as a ligand. Folic acid is capable of binding to cell-surface folate receptors with high affinity. These receptors have become important molecular targets for cancer research as they are overexpressed in numerous cancers despite being expressed at low levels in normal tissues. Employing a post-column copper-catalyzed alkyne-azide cycloaddition (CuAAC), we report the synthesis of siRNAs bearing folic acid modifications at different positions within the sense strand. In the absence of a transfection carrier, these siRNAs were selectively taken up by cancer cells expressing folate receptors. We show that centrally modified folic acid-siRNAs display enhanced gene-silencing activity against an exogenous gene target (∼80% knockdown after 0.75 µM treatment) and low cytotoxicity. In addition, these siRNAs achieved potent dose-dependent knockdown of endogenous Bcl-2, an important anti-apoptotic gene.

Effective carrier-free gene-silencing activity of cholesterol-modified siRNAs.

The use of short interfering RNAs (siRNAs) as therapeutics holds great promise, but chemical modifications must first be employed to improve their pharmacokinetic properties. This study evaluates the in vitro cellular uptake and knock-down efficacy of cholesterol-modified triazole-linked siRNAs targeting firefly luciferase in the absence of a transfection carrier. These siRNAs displayed low cytotoxicity and excellent dose-dependent knockdown in HeLa cells in the 500 to 3000 nM concentration range, with a 70-80% reduction in firefly luciferase activity. Our results indicate that this modification is compatible with the RNA interference pathway, and is less cytotoxic and more effective than a commercially-available triethylene glycol (TEG) cholesterol modification.

Efficient synthesis of the GABAA receptor agonist trans-4-aminocrotonic acid (TACA).

Investigations into the pharmacology of different types of cys-loop GABA receptor have relied for years on the chemical modification of GABA-like compounds. The GABA metabolite GABOB is an attractive molecule to modify due to its convenient chemical structure. In the process of developing new GABA-mimic compounds from GABOB as a starting compound three small molecule GABA derivatives were synthesized using a variety of chemical transformations. Amongst these, a new and reliable method to synthesize TACA (trans-4-aminocrotonic acid) is reported.