SiRNA labeling and delivery into cells

Abstract

Small interfering RNAs (siRNAs) are short double-stranded RNA molecules, ranging 20-25 base pairs in length. They are known to play a crucial role in gene regulation by modulating RNA interference (RNAi) pathway. The ability to control gene expression has made siRNAs a potential therapeutic target for diverse diseases. Delivering and visualizing the siRNAs, both in-vitro and in-vivo, to gain insights into their uptake and understanding the mechanism of action requires development of labeling technology that can provide detection of small amounts of siRNAs. Fluorescent labeling of siRNA enables the visualization, quantification, and tracking of siRNAs within cells and tissues. Monitoring the delivery, uptake, and distribution of siRNAs by detecting the emission from fluorescent tags provides a convenient solution. Additionally, fluorescently labeled siRNAs can also validate their targeting specificity. Real-time monitoring of gene silencing effects becomes possible, facilitating assessments of siRNA pharmacokinetics studies. The fairly simple method of labeling siRNAs and delivering them to the cells as described in this study provides an excellent solution for fundamental research on siRNAs and their therapeutic applications through RNAi pathway.

Introduction

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The advent of precision medicine enabled by progressions in genome sequencing and bioinformatics, in the last decade marked a significant milestone in the field of healthcare. This personalized approach for treating several diseases maximizes the treatment efficacy and minimizes the toxic off-target effects. An upcoming field within precision medicine is harnessing the RNA interference pathway (also known as RNAi), modulated by siRNAs [1]. Improvements in nucleic acid design and synthesis technology has enabled the development of robust, rapid, and cost-effective production of siRNA sequences, as compared to small molecule and antibody drugs, being traditionally used in disease therapeutics [2]. The possibility of designing siRNA sequences targeting the undruggable sequences/loci, could be a lucrative treatment approach. As a result, the use of RNAi technology and siRNAs has attracted much attention in the recent past. The first siRNA therapeutic drug to be approved by US FDA, was ONPATTRO™ (Patisiran), developed by Alnylam Pharmaceuticals, Inc. in 2018 for treating hereditary ATTR (hATTR) Amyloidosis with Polyneuropathy [3,4]. As of today, five siRNA drugs have been approved by the US FDA and few more are under clinical trials [1, 5].

To accelerate the pre-clinical studies aimed at improving and better understanding siRNAs in disease therapy requires development of tools for delivering and visualizing the siRNAs. Fluorescent labeling/tagging of siRNA sequence has been very challenging due to the lack of reactive moieties in nucleic acid molecules. Thymine and Guanosine have been often explored for nucleic acid conjugations, e.g., photo-crosslink (to thymine by psoralens) or bromination/Ulysis labeling of guanosine. However, these existing conjugation techniques are either tedious, ineffective or require stringent conditions associated with low yields and are thus not suitable for routine lab use. Development of a labeling reagent that readily reacts with the N7 of guanine to form a stable covalent bond provides a simple, time-saving and cost-effective solution to this problem. The separation of the labeled nucleic acids from the unreacted dye can be accomplished with a simple ethanol precipitation. The resulting labeled RNA probes have bright and stable fluorescence that can be easily detected with the Cy5 filter set. Delivering these modified siRNA to the cells for achieving gene knockdown with real-time monitoring of gene silencing effects can be achieved by transfecting the cells with easy-to-use transfection reagents, facilitating assessments of siRNA knockdown studies. The simpler approach of fluorescently labeling siRNAs using our Helixyte™ iFluor® 647 Nucleic Acid Labeling Dye and delivering them to the cells using Transfectamine 7000 siRNA transfection a-reagent as described in the current study, can help accelerate the fundamental research on siRNAs.

Materials and Methods

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Cell culture

HeLa cells were cultured and maintained in DMEM with 10% Fetal Bovine Serum (FBS) and 1% antibiotic (100 U/mL Penicillin and 100 μg/mL Streptomycin), under a humidified atmosphere at 37°C and 5% CO2.

Labeling siRNAs

ON-TARGET plus GAPD Control siRNA was purchased from Horizon Discovery. This was labeled in-house with 100 or 200 μM Cy5 using the Helixyte™ nucleic acid labeling dye from AAT Bioquest, followed by purification and alcohol precipitation of labeled siRNA. Spectral peaks were analyzed by estimating the absorbance of the labeled siRNA and by agarose gel electrophoresis.

Transfection of labeled siRNA

Cells were seeded in a 96-well plate 6 hours prior to transfection. At the time of transfection, cells were grown around 80% confluency in the plate. (Note: Higher confluency is recommended for data analysis at 24-48 hours and lower confluency for 48-72 hours). For transfecting the siRNA into the HeLa cells, Transfectamine 7000 siRNA transfection reagent was used. Cell culture medium was replaced with 80 μL of low serum medium. 50nM of Cy5 labeled siRNA was prepared in 10 μL of low-serum medium and 0.15 μL of Transfectamine 7000 was mixed in 10 μL medium. The siRNA and reagent were mixed by gentle vortexing and letting the mixture rest at RT for 10 mins. This mix was added to cells and incubated overnight. The uptake of labeled siRNA was monitored under the Cy5 filter set of fluorescence microscope.

Results

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Absorption spectra of Cy5 labeled siRNA

ON-TARGETplus GAPD Control siRNA was labeled with 100 or 200 μM of Cy5 using the Helixyte™ nucleic acid labeling kit. This was followed by purification and alcohol precipitation of labeled siRNA. Spectral peaks were analyzed by estimating the absorbance of the labeled siRNA and by agarose gel electrophoresis. Both the labeling concentrations gave good absorbance spectra at 647nm. The Base:Dye ratio for 100 μM Cy5 labeled siRNA is 2.9 and for 200 μM Cy5 labeled siRNA is 0.8.

Agarose gel electrophoresis of Cy5 labeled siRNA

100ng each of siRNA (unlabeled), Cy5*siRNA (100 μM) and Cy5*siRNA (200 μM) were run on in-house casted 2% agarose gel. The gel was stained with CyberOrange dye and observed under Sypro Ruby and Cy5 filters. The unlabeled siRNA and Cy5 labeled siRNA are observed to be migrating together. The labeled siRNAs are showing up under Cy5 filter (Red) unlike the unlabeled counterpart (Blue).

Transfection of Cy5 labeled siRNA into cells

50nM of Cy5 labeled siRNA (Labeled with 100 μM dye) was transfected into HeLa cells using Transfectamine 7000 siRNA transfection reagent. The cells were monitored at 24, 48 and 72 hours after transfection, under the Cy5 filter set of fluorescence microscope to study the uptake of labeled siRNA by HeLa cells. The transfected HeLa cells are showing presence of Cy5 labeled siRNA as early as 24 hours after transfecting the cells, and this uptake is more pronounced at 72 hours.

Conclusions

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In this assay wise letter, we have addressed two key issues, one is labeling the siRNA for better in-vitro visualization using the Helixyte™ nucleic acid labeling dye, and the other is transfecting siRNAs into the cells using the Transfectamine 7000 siRNA transfection reagent. We have successfully shown that siRNAs can be labeled and transfected into cells using the Transfectamine 7000 transfection reagent.

References

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