Complete, gene-specific siRNA libraries: production and expression in mammalian cells

Abstract

Short interfering RNAs (siRNAs) are widely used to silence the expression of specific genes. Current practice for designing effective siRNAs is to use algorithms based on sequence-efficacy correlations; however, there are many highly effective sequences that these algorithms do not anticipate. To ensure that the best siRNAs are identified, all possible gene-specific siRNA sequences of appropriate lengths should be screened in cell culture. Synthesizing and testing all such sequences individually is costly. A potentially much easier alternative is to prepare a mixture of all these sequences (a gene-specific library), express them in cells, select cells having the desired phenotype, and identify the siRNA contained within the selected cells. Here we describe two new methods for preparing and expressing such libraries. The first uses cloned Dicer or RNase III to digest gene-specific RNA duplexes to siRNAs, which are then converted to the corresponding DNA sequences by attaching RNA primers and performing reverse transcription-PCR. The second method involves partial DNase I digestion of gene-specific DNA, purification of a 20-30-bp fraction, and amplification by attaching DNA adapters followed by PCR. DNA libraries specific for TNF-alpha, DsRed, and part of the hepatitis C virus genome, generated by methods, were inserted into siRNA expression vectors between convergent human U6 and H1 promoters. Randomly selected clones from each library together with vectors expressing the corresponding target genes were cotransfected into 293FT cells and assayed for target gene inhibition. About 10%-20% of siRNAs represented in these libraries show significant inhibition of their target genes. Most of these inhibitory sequences are not predicted by existing algorithms.

FIGURE 1.

Preparation of gene-specific siRNA libraries by Dicer fragmentation of double-stranded RNA. (A) The general scheme. The double-stranded RNA target is digested by Dicer (or RNase III) to produce 21–22-bp siRNAs. In two subsequent ligation steps, single-stranded RNA adapters are attached to the 3′ and 5′ ends of each fragment by T4 RNA ligase. The products of ligation are reverse transcribed and PCR amplified using the oligonucleotides attached to the gene-derived sequences as primer-binding sites. The resulting PCR products are cut with appropriate restriction enzymes and cloned into the siRNA expression vector pU6/H1-coh (see Fig. 3 ▶). (B) Sequencing results for the randomly selected clones from the TNF-specific library.

FIGURE 2.

Preparation of gene-specific siRNA libraries by DNase I fragmentation of double-stranded DNA. (A) The general scheme. The double-stranded DNA target is digested by DNase I in the presence of Mn²⁺ ions, and the fraction containing 20–30-bp fragments is gel-purified. Next, double-stranded DNA adapters are attached to 3′ and 5′ ends by T4 DNA ligase, and the resulting fragments are amplified by PCR. Further, fragments are cut with appropriate restriction enzymes and cloned into pU6/H1-coh (see Fig. 3 ▶). (B) Sequencing results for the randomly selected clones from the DsRed-specific library.

FIGURE 3.

Scheme for expression of siRNA libraries from opposing pol III promoters. (A) U6/H1 expression cassette used for cloning of cohesive-ended fragments (pU6/H1-coh; modified from Zheng et al. 2004). (B) The U6/H1 expression cassette allowing blunt-end cloning of siRNA library inserts (pU6/H1-blunt; see Materials and Methods).

FIGURE 4.

Silencing ability of species randomly selected from the TNF-specific siRNA library produced by the Dicer method. (A) 293FT cells were cotransfected with randomly chosen clones, a TNF expression vector, and pSEAP using Lipofectamine 2000 (Invitrogen). TNF was assayed by ELISA and SEAP by a colorimetric assay 48 h post-transfection. The inhibition by each siRNA is shown, normalized to the SEAP control target. Rationally designed control shRNAs targeting TNF (shRNA-TNF-229) and DsRed (shRNA-DsRed-2) were expressed from pU6. Rationally designed control siRNAs targeting TNF (siRNA-TNF-229) and DsRed (siRNA-DsRed-2) were expressed from pU6/H1. (B) Representative sequences of the assayed clones were classified into three groups depending on reduction in TNF expression: effective (>60% inhibition), intermediate (20%–60%), and ineffective (<20%).

FIGURE 5.

Silencing ability of species randomly selected from the DsRed-specific siRNA library produced by the DNase I method. (A) Randomly chosen clones were cotransfected with DsRed expression vector into 293FT cells with Lipofectamine 2000 (Invitrogen). DsRed protein levels were quantified by flow cytometry 48 h after transfection. Cells were also imaged by fluorescence microscopy. The amount of inhibition of each siRNA was normalized to the pU6/H1 empty vector. Rationally designed control siRNAs targeting DsRed (siRNA-DsRed-2) TNF, (siRNA-TNF-229), and eGFP (siRNA-eGFP) were expressed from pU6/H1. Rationally designed control shRNA targeting DsRed (shRNA-DsRed-2) was expressed from pU6. (B) Representative sequences of the assayed clones, classified as in Figure 4 ▶.