SARS-CoV-2 viral titer measurements in Ontario, Canada wastewaters throughout the COVID-19 pandemic.

During the COVID-19 pandemic, the Province of Ontario, Canada, launched a wastewater surveillance program to monitor SARS-CoV-2, inspired by the early work and successful forecasts of COVID-19 waves in the city of Ottawa, Ontario. This manuscript presents a dataset from January 1, 2021, to March 31, 2023, with RT-qPCR results for SARS-CoV-2 genes and PMMoV from 107 sites across all 34 public health units in Ontario, covering 72% of the province's and 26.2% of Canada's population. Sampling occurred 2-7 times weekly, including geographical coordinates, serviced populations, physico-chemical water characteristics, and flowrates. In doing so, this manuscript ensures data availability and metadata preservation to support future research and epidemic preparedness through detailed analyses and modeling. The dataset has been crucial for public health in tracking disease locally, especially with the rise of the Omicron variant and the decline in clinical testing, highlighting wastewater-based surveillance's role in estimating disease incidence in Ontario.

The role of catchment population size, data normalization, and chronology of public health interventions on wastewater-based COVID-19 viral trends.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic presented the most challenging global crisis in recent times. A pandemic caused by a novel pathogen such as SARS-CoV-2 necessitated the development of innovative techniques for the monitoring and surveillance of COVID-19 infections within communities. Wastewater surveillance (WWS) is recognized as a non-invasive, cost-effective, and valuable epidemiological tool to monitor the prevalence of COVID-19 infections in communities. Seven municipal wastewater sampling sites representing distinct sewershed communities were selected for the surveillance of the SARS-CoV-2 virus in Durham Region, Ontario, Canada over 8 months from March 2021 to October 2021. Viral RNA fragments of SARS-CoV-2 and the normalization target pepper mild mottle virus (PMMoV) were concentrated from wastewater influent using the PEG/NaCl superspeed centrifugation method and quantified using RT-qPCR. Strong significant correlations (Spearman's rs = 0.749 to 0.862, P < 0.001) were observed between SARS-CoV-2 gene copies/mL of wastewater and clinical cases reported in each delineated sewershed by onset date. Although raw wastewater offered higher correlation coefficients with clinical cases by onset date compared to PMMoV normalized data, only one site had a statistically significantly higher Spearman's correlation coefficient value for raw data than normalized data. Implementation of community stay-at-home orders and vaccinations over the course of the study period in 2021 were found to strongly correspond to decreasing SARS-CoV-2 wastewater trends in the wastewater treatment plants and upstream pumping stations.

Cover Feature: Direct Conjugation of Gallium-(III)-Corroles to Short Interfering RNA(siRNA) Providing Real-Time siRNA Imaging and Gene Silencing (ChemPlusChem 6/2024).

Invited for this month's cover is the group of Jean-Paul Desaulniers at Ontario Tech University. The cover picture shows the successful conjugation of a GaIII-corrole to an siRNA to enable live cell imaging. Read the full text of the article at 10.1002/cplu.202400084.

Direct Conjugation of Gallium-(III)-Corroles to Short Interfering RNA(siRNA) Providing Real-Time siRNA Imaging and Gene Silencing.

Discovering new modifications for oligonucleotide therapeutics is essential for expanding its application to new targets and diseases. In this project, we focus on conjugating metaled ligands to short interfering RNAs (siRNAs) to investigate robust and simple conjugation methods for adding new properties such as real-time imaging to the siRNA. Here we report the chemical synthesis of novel Ga-(III)-corroles for their direct conjugation to siRNAs. Ga-(III)-corrole-siRNAs showed promising results when evaluated for gene silencing and live cell imaging. The knockdown activity of the firefly luciferase reporter gene was measured to evaluate gene silencing activity. Gene silencing studies from two 5'-Ga-(III)-labeled-siRNAs exhibited dose-dependent knockdown with IC50s of 812.7 and 451.4 pM, which is comparable to wild-type (IC50=439.7 pM) in the absence of red light, and IC50s of 562.9 and 354.5 pM, which is also comparable to wild-type (IC50=337.4 pM), in the presence of red light. In addition, imaging studies with Ga-(III)-corrole-modified siRNAs showed intense fluorescence in HeLa cells, highlighting that the Ga-(III)-corrole modification is an effective fluorophore for siRNA tracing and imaging. Moreover, the photodynamic activity of free base corrole vs the Ga-(III)-corrole was evaluated. Results show an increase of light cytotoxicity of the corrole ligand upon the addition of Ga-(III); however, no phototoxicity was observed when Ga-(III) ligands were linked to siRNA. In conclusion, Ga-(III)-corrole-siRNAs show promising results for applications in simultaneous real-time imaging and gene silencing.

Synthesis of an Ortho-Functionalized Tetrafluorinated Azobenzene Phosphoramidite for Incorporation into a Tetrafluorinated Azobenzene-Containing siRNA for Photocontrolled Gene Silencing.

This article presents the detailed synthesis and characterization protocols for an ortho-functionalized tetrafluorinated azobenzene containing siRNA, which has photoswitchable properties. To design this tetrafluorinated azobenzene scaffold, several synthetic steps are performed to generate a symmetrical tetrafluorinated azobenzene diol. This diol is treated with dimethoxytrityl chloride (DMT-Cl) to protect one of the alcohols. Next, the DMT-protected tetrafluorinated monoalcohol is phosphitylated to afford the DMT-phosphoramidite building block used for solid-phase synthesis. This paper also contains the detailed biophysical characterization, biological testing, and photo-switching protocols of an ortho-functionalized fluorinated azobenzene containing siRNA (F-siRNA), which has photoswitchable properties that can be controlled with visible light. First, the F-siRNA was characterized by annealing the sense and antisense strands together and then measuring the circular dichroism (CD) profile and melting temperature (Tm ) of the duplexes. Second, biological testing of the F-siRNA is performed in cell culture to determine their gene silencing efficacy. Finally, their gene-silencing activities are measured after exposure to green light to inactivate the F-siRNA, followed by blue light, which reactivates the F-siRNA. The F-siRNA can be kept inactive for up to 72 hr and reactivated at any time within this 72-hr window. © 2023 Wiley Periodicals LLC.

An alternative method for monitoring and interpreting influenza A in communities using wastewater surveillance.

Seasonal influenza is an annual public health challenge that strains healthcare systems, yet population-level prevalence remains under-reported using standard clinical surveillance methods. Wastewater surveillance (WWS) of influenza A can allow for reliable flu surveillance within a community by leveraging existing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) WWS networks regardless of the sample type (primary sludge vs. primary influent) using an RT-qPCR-based viral RNA detection method for both targets. Additionally, current influenza A outbreaks disproportionately affect the pediatric population. In this study, we show the utility of interpreting influenza A WWS data with elementary student absenteeism due to illness to selectively interpret disease spread in the pediatric population. Our results show that the highest statistically significant correlation (Rs = 0.96, p = 0.011) occurred between influenza A WWS data and elementary school absences due to illness. This correlation coefficient is notably higher than the correlations observed between influenza A WWS data and influenza A clinical case data (Rs = 0.79, p = 0.036). This method can be combined with a suite of pathogen data from wastewater to provide a robust system for determining the causative agents of diseases that are strongly symptomatic in children to infer pediatric outbreaks within communities.

Synthesis of Dinucleotide Non-Symmetrical Triester Phosphate Phosphoramidites and Their Incorporation Within Oligonucleotides.

In this protocol article, the synthesis of dinucleotide non-symmetrical triester phosphate phosphoramidites will be highlighted. Specifically, we use a selective transesterification starting with tris(2,2,2-trifluoroethyl) phosphate to afford a dinucleotide derivative phosphate ester. Substitution of the final trifluoroethyl group with various alcohols affords a dinucleotide triester phosphate with a hydrophobic group, which can then be deprotected and converted to a phosphoramidite for incorporation within oligonucleotides. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of a DMT- and TBS-protected unsymmetrical dinucleotide Basic Protocol 2: Synthesis of a DMT-protected unsymmetrical dinucleotide phosphotriester monoalcohols Basic Protocol 3: Synthesis of DMT-protected phenylethyl phosphotriester dinucleotide phosphoramidites Basic Protocol 4: Synthesis, purification, and characterization of RNAs containing triester phosphate modifications.

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.

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

RNA interference (RNAi) is a natural cellular process that silences the expression of target genes in a sequence-specific way by mediating targeted mRNA degradation. One of the main challenges in RNAi research is developing an effective career-free delivery system and targeting cells in the central nervous system (CNS). Recently, lipid-conjugated systems involving fatty acids have shown promising potential as safe and effective delivery systems of oligonucleotides to CNS cells due to their hydrophobic tails and interactions with the cell's hydrophobic membrane. Therefore, in this study, we are interested in creating career-free siRNA therapeutics for potential applications in drug delivery to the CNS. Here we explore different synthetic pathways of conjugating sphingolipids containing long-carbon chains to siRNA and assess their effectiveness as career-free delivery systems. In this project, a library of sphingosine-modified siRNAs was created, and their gene-silencing effect was evaluated in both the presence and absence of a transfection carrier. siRNAs modified with one or two sphingosine moieties resulted in dose-dependent gene knockdown while demonstrating promising results for their use as carrier-free agents. The IC50 values of single-modified siRNAs ranged from 49.9 nM to 670.7 nM, whereas double-modified siRNAs had IC50 values in the range of 49.9 nM to 66.4 nM. In conclusion, sphingosine-modified siRNAs show promising results in advancing carrier-free siRNA therapeutics.

Synthesis of 5'-fluorophosphate-modified short-interfering RNAs.

We have developed an improved scheme for the synthesis of a mono-fluorinated phosphoramidite for the 5'-modification of nucleic acids using standard phosphoramidite chemistry. We describe the first report of a phosphofluoridate modified siRNA strand and have evaluated C18 HPLC for purification of modified strands from unreacted siRNA strands. Lastly, the biological activity of the high purity siRNA strands, when placed on the sense and/or antisense strand, was evaluated to assess the impact of 5' phosphofluoridate modifications on siRNA activity.

Synthesis of Ortho-Functionalized Tetrachlorinated Azobenzene Phosphoramidites for Incorporation Into siRNAzos for Photocontrolled Gene Silencing.

This paper contains the detailed synthesis and characterization protocols of ortho-functionalized tetrachlorinated azobenzene-containing small interfering RNAs (siRNAs), which have photoswitchable properties effectively controlled with visible light. To design this tetrachlorinated azobenzene scaffold, a late-stage chlorination with N-chlorosuccinimide and palladium is used. Next, a single hydroxyl group from the tetrachlorinated azobenzene is protected with a 4,4'-dimethoxytrityl (DMT) group, followed by phosphitylation with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite. These phosphoramidite monomers are compatible with automated solid-phase oligonucleotide synthesis to generate tetrachlorinated azobenzene-containing oligonucleotides. This paper also contains the detailed biophysical characterization, biological testing, and photo-switching protocols of ortho-functionalized chlorinated azobenzene-containing siRNAs (Cl-siRNAzos), which have photoswitchable properties that can be controlled with visible light. First, the Cl-siRNAzos are characterized by annealing the sense and antisense strands together and then measuring the circular dichroism (CD) profile, and the melting temperatures (Tm ) of the duplexes. Secondly, the biological testing of the Cl-siRNAzos in cell culture is done to determine their gene silencing efficacy. Finally, their gene-silencing activities are measured after exposure to red light in order to inactivate the Cl-siRNAzo, and then either violet light or infrared thermal relaxation is deployed, which re-activates the Cl-siRNAzo. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 4,4'-bis(hydroxyethyl) ortho-functionalized tetrachlorinated azobenzene phosphoramidite (5) Basic Protocol 2: Synthesis, purification, and characterization of siRNAs containing ortho-functionalized tetrachlorinated azobenzene Basic Protocol 3: Gene-silencing evaluation of ortho-functionalized tetrachlorinated azobenzene using firefly luciferase.

Synthesis, Derivatization and Photochemical Control of an ortho-Functionalized Tetrafluorinated Azobenzene-Modified siRNA.

We report the synthesis of an ortho-functionalized tetrafluorinated azobenzene phosphoramidite for its site-specific incorporation into RNA. The tetrafluorinated azobenzene is embedded within the antisense strand of an siRNA duplex to form an ortho-functionalized tetrafluorinated azobenzene-containing siRNA (F-siRNAzo). The F-siRNAzo is inactivated via trans to cis conversion with green light (530 nm), and reactivated with blue light (470 nm) via cis to trans conversion in cell culture. The long half-life and stability of the tetrafluorinated azobenzene unit allows for reversible control of the F-siRNAzo in cell culture for up 72 hours.

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.

Detection of SARS-CoV-2 Proteins in Wastewater Samples by Mass Spectrometry.

The recent COVID-19 pandemic overwhelmed the health system worldwide, and there was a need to track outbreaks and try to use this information as an early warning system. Wastewater-based epidemiology (WBE) enabled detection of the SARS-CoV-2 virus in wastewater treatment plant influents. Until now, the most used technique for this detection has been the quantitative polymerase chain reaction (qPCR)-based quantification of SARS-CoV-2 RNA. This study proposes a mass spectrometry (MS)-based method that detected specific SARS-CoV-2 proteins in wastewater, 5 and 6 days ahead of the case data for two municipalities. We identified unique peptides of eight proteins related to the SARS-CoV-2 virus and COVID-19 infection. We detected the nonstructural protein (NSP) pp1ab (transcribed after host cell infection) most frequently in all of the samples. As a result, we suspect that in the active cases of COVID-19, the pp1ab protein is present in high abundance in the urine and feces and that this protein could be used as an alternative biomarker. These data were collected before mass vaccination occurred in the population.

A dual therapeutic system based on corrole-siRNA conjugates.

Corrole molecules are a new generation of photosensitizers (PS) due to their ease of tunability for different medical applications. Their ability to initiate cellular death using a wide range of non-toxic wavelengths allows for the creation of non-invasive treatments. This work focuses on creating potent and non-invasive treatments by advancing siRNA therapeutics by directly conjugating siRNAs with the photosensitizer, corrole. Combining gene silencing with photodynamic therapy (PDT) creates a non-invasive dual therapy system. Basic synthetic designs were explored to create novel corrole-phosphoramidites and from these, a small library of corrole-functionalized short interfering RNAs (corrole-siRNAs) were synthesized. Corrole-siRNA conjugates showed promising results when evaluated for gene silencing and PDT therapy in vitro. Gene silencing effects were evaluated in cells by measuring the knockdown activity of the firefly luciferase reporter gene. Gene silencing studies from four siRNAs showed promising dose dependent knockdown with IC50s of 387.8, 77.8, 60.0, and 49.4 pM in the absence of red light, and 101.0, 57.2, 55.3, and 23.8 pM in the presence of red light. Furthermore, PDT showed approximately a 50% decrease in cell viability for red-light irradiated cells treated with corrole-siRNAs, demonstrating the effective role of corrole to act as a photosensitizer while still maintaining robust siRNA activity. In conclusion, corrole-siRNAs show a promising path for developing novel siRNA combination therapy.

Pasteurization, storage conditions and viral concentration methods influence RT-qPCR detection of SARS-CoV-2 RNA in wastewater.

The COVID-19 pandemic presents many public health challenges including the tracking of infected individuals from local to regional scales. Wastewater surveillance of viral RNA has emerged as a complementary approach to track and monitor the presence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus in a variety of communities of different land use and population size. In the present study, we investigate how five different parameters (pasteurization, storage temperature, storage time, polyethylene glycol (PEG) concentration, and pellet mass) affect the detection of the SARS-CoV-2 N gene and fecal abundance indicator pepper mild mottle virus (PMMoV) gene. Pre-treatment of 24-h composite wastewater samples (n = 14) by pasteurization at 60 °C resulted in a significant reduction of total RNA concentration and copies of the SARS-CoV-2 N gene copies/L (paired Student's t-test, P < 0.05). Comparing the wastewater samples collected from 6 wastewater treatment plants (WWTPs) for a storage period of 7 and 14 days at 4 °C, -20 °C and -80 °C, demonstrated a decrease in SARS-CoV-2 N gene copies/L when samples were stored for 14 days at -20 °C. Polyethylene glycol-NaCl for purification and concentration of viral particles from the wastewater samples demonstrated that a short PEG incubation of 2 h during centrifugation at 4 °C was sufficient for the consistent detection of the SARS-CoV-2 N gene from a 30 mL sample volume. Combined, this paper presents method recommendations for developing a reliable, accurate, sensitive, and reproducible estimation of the SARS-CoV-2 virus in diverse domestic wastewater samples.

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.

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.

Chemical strategies for strand selection in short-interfering RNAs.

Therapeutic small interfering RNAs (siRNAs) are double stranded RNAs capable of potent and specific gene silencing through activation of the RNA interference (RNAi) pathway. The potential of siRNA drugs has recently been highlighted by the approval of multiple siRNA therapeutics. These successes relied heavily on chemically modified nucleic acids and their impact on stability, delivery, potency, and off-target effects. Despite remarkable progress, clinical trials still face failure due to off-target effects such as off-target gene dysregulation. Each siRNA strand can downregulate numerous gene targets while also contributing towards saturation of the RNAi machinery, leading to the upregulation of miRNA-repressed genes. Eliminating sense strand uptake effectively reduces off-target gene silencing and helps limit the disruption to endogenous regulatory mechanisms. Therefore, our understanding of strand selection has a direct impact on the success of future siRNA therapeutics. In this review, the approaches used to improve strand uptake are discussed and effective methods are summarized.

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.