A magnetic bead-integrated chip for the large scale manufacture of normalized esiRNAs

Abstract

The chemically-synthesized siRNA duplex has become a powerful and widely used tool for RNAi loss-of-function studies, but suffers from a high off-target effect problem. Recently, endoribonulease-prepared siRNA (esiRNA) has been shown to be an attractive alternative due to its lower off-target effect and cost effectiveness. However, the current manufacturing method for esiRNA is complicated, mainly in regards to purification and normalization on a large-scale level. In this study, we present a magnetic bead-integrated chip that can immobilize amplification or transcription products on beads and accomplish transcription, digestion, normalization and purification in a robust and convenient manner. This chip is equipped to manufacture ready-to-use esiRNAs on a large-scale level. Silencing specificity and efficiency of these esiRNAs were validated at the transcriptional, translational and functional levels. Manufacture of several normalized esiRNAs in a single well, including those silencing PARP1 and BRCA1, was successfully achieved, and the esiRNAs were subsequently utilized to effectively investigate their synergistic effect on cell viability. A small esiRNA library targeting 68 tyrosine kinase genes was constructed for a loss-of-function study, and four genes were identified in regulating the migration capability of Hela cells. We believe that this approach provides a more robust and cost-effective choice for manufacturing esiRNAs than current approaches, and therefore these heterogeneous RNA strands may have utility in most intensive and extensive applications.

Figure 1. Manufacture of esiRNA by means of a magnetic bead-integrated chip.

A. The schematic diagram shows the chip composed of a microwell array and magnetic beads coated with streptavidin. B. Large-scale manufacture of esiRNAs can be divided into three steps: target amplification and immobilization, transcription, and enzymatic digestion. C. Confirmation of PCR reactions using biotinylated or non-biotinylated primers. Amplification products (left), transcription products (middle), or esiRNA products (right) are detected only when biotinylated DNA primers are used. B: biotinylated primers.

Figure 2. The normalization of esiRNA products.

A. A range of initial amounts of DNA template were assayed (100, 10, 1, and 0.1 ng, each in 5 µL in volume). After the immobilization and transcription steps, the variation of transcription products was within 20%. The final esiRNA products had a variation of less than 10% when the initial amount of DNA templates was in the range of 0.1–100 ng. B. Eight esiRNA products were manufactured in parallel. The standard deviation among these eight products was approximately 3%.

Figure 3. The silencing specificity and efficiency of esiRNAs. A.

Fifteen GFP esiRNAs generated in parallel potently inhibited the fluorescent signal in cells cotransfected with GFP-encoding vectors; in contrast, five PLAU esiRNAs had no effect on the fluorescent intensity compared to control experiments. B. & C. Quantitative analysis of the silencing efficiency of esiRNA products. The qRT-PCR results showed that esiRNAs manufactured on the magnetic beads can efficiently inhibit expression levels of the respective genes. A western blot assay showed that esiRNAs targeting TP53, TGFB1, PLAU, or TGFBR2 inhibited therespective protein expression levels by up to approximately 50%.

Figure 4. Synergistic effects of esiRNAs.

A. The simultaneous production of two normalized esiRNAs in one well. B. The qRT-PCR results showed that the mixture of PARP1 and BRCA1 esiRNAs could effectively silence the expression of both genes. C. & D. The MTS assay and cell survival assays showed the synergistic effect of PARP1 and BRCA1 genes.

Figure 5. Screen and validation of tyrosine kinase genes capable of regulating the cell migration. A.

Self-assembled cell microarray screen of genes capable of regulating the migration of Hela cells by use of esiRNA tyrosine kinase library. B. Validation of the esiRNA screen results by use of siRNAs and transwell assay.