Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers.
TRUE gene silencing (termed after tRNase ZL–utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes an artificial small guide RNA (sgRNA) to guide tRNA 3′ processing endoribonuclease, tRNase ZL, to recognize a target RNA. sgRNAs can be taken up by cells without any transfection reagents and can downregulate their target RNA levels and/or induce apoptosis in human cancer cells. We have screened an sgRNA library containing 156 heptamer-type sgRNAs for the effect on viability of human myeloma and leukemia cells, and found that 20 of them can efficiently induce apoptosis in at least one of the cancer cell lines. Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers. The protocol includes how to construct the sgRNA library, how to assess the effect of each sgRNA on cell viability, and how to further evaluate the effective sgRNAs by flow cytometry. Around 2,000 hits would be expected to be obtained by screening the full-scale sgRNA library composed of 16,384 heptamers.
TRUE gene silencing (termed after tRNase ZL–utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies1. This technology has been developed based on properties of tRNase ZL (or 3′ tRNase), tRNA 3′ processing endoribonuclease: it can cleave any target RNA at any expected site under the direction of an artificial small guide RNA (sgRNA) by recognizing a pre-tRNA-like or micro-pre-tRNA-like structure formed between the target RNA and the sgRNA2–9. sgRNA, which is usually 7-31 nt in length, is categorized into four groups, 5′-half-tRNA, heptamer RNA, 14-nt linear RNA, and hook RNA.
We have demonstrated the efficacy of TRUE gene silencing by introducing various artificially-designed sgRNAs into living cells10–15. We have also shown that sgRNAs can be taken up by cells without any transfection reagents and can downregulate their target RNA levels and/or induce apoptosis in human cancer cells16–18. TRUE gene silencing appears to work on the intracellular and intercellular gene regulatory network system via tRNase ZL and natural sgRNAs19–21.
We have constructed an sgRNA library containing 156 heptamer-type sgRNAs to search for potential therapeutic heptamer-type sgRNAs for blood cancers22. We have screened it for the effect on viability of human myeloma and leukemia cells, and found that 20 of them can efficiently induce apoptosis in at least one of the cancer cell lines. And 4 of them have been shown to reduce tumor growth rates in mouse xenograft experiments.
Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers. The protocol includes how to construct the sgRNA library, how to assess the effect of each sgRNA on cell viability, and how to further evaluate the effective sgRNAs by flow cytometry. Analysis for sgRNA's cellular target RNAs and evaluation of effective sgRNAs in mouse xenograft models can be performed according to conventional methods17,22, although these are not included in this protocol.
1. Construction of a Heptamer-type sgRNA Library
2. Screening of the sgRNA Library for Cell Viability
3. Evaluation of Effective sgRNAs by Flow Cytometry
The six heptamer-type sgRNAs 5′-AUCUUCA-3′ (H1885), 5′-ACACACA-3′ (H3277), 5′-GGGGGCG-3′ (H10927), 5′-GGGGCCC-3′ (H10944), 5′-GCCCCCG-3′ (H12287), and 5′-CACCAGC-3′ (H13260) were chemically synthesized as 2′-O-methyl RNA containing 5′- and 3′-phosphates.
These sgRNAs were examined for the effects on viability of a human leukemia cell line, HL60, and a human myeloma cell line, RPMI-8226. Representative results of the cell viability assays are shown in Figure 1. The four sgRNAs H1885, H3277, H10927, and H13260 were very effective and reduced the viability of HL60 and RPMI-8226 cells by >80% and >70%, respectively. The heptamers H3277 and H10927 were also tested for the effects on non-cancerous HEK293 cell viability, and have been shown to hardly affect the cell viability22.
The flow cytometry with annexin V and 7AAD double staining was performed for HL60 cells treated with the heptamer H1885, H3277, H10927, or H13260. In each sgRNA, a total cell number (56-95%) in early and late apoptotic stages was much higher than that (4-6%) in mock (Figure 2). These observations suggest that the reduction of HL60 cell viability is due to apoptosis.
Figure 1. Cell viability assays. Cell viability was measured 72 hr after HL60 cells (A) or RPMI-8226 cells (B) were cultured in the absence or presence of 1 µM of the naked heptamer-type sgRNA H1885, H3277, H10927, H10944, H12287, or H13260. The relative living cell numbers in the absence of sgRNAs are adjusted to 100. Error bars indicate SD (n = 3). *, P < 0.002. Reprinted from Leukemia Research, Vol. 38, Takahashi M. et al., Screening of a heptamer-type sgRNA library for potential therapeutic agents against hematological malignancies, pp 808-815, Fig. 1, Copyright (2014), with permission from Elsevier. Please click here to view a larger version of this figure.
Figure 2. Flow cytometry. With respect to HL60 cells, flow cytometry was performed with annexin V and 7AAD double staining. The cells were analyzed 72 hr after cultured in the absence or presence of 1 µM of the naked heptamer-type sgRNA H1885 (A), H3277 (A), H13260 (A), or H10927 (B). Reprinted from Leukemia Research, Vol. 38, Takahashi M. et al., Screening of a heptamer-type sgRNA library for potential therapeutic agents against hematological malignancies, pp 808-815, Fig. 2, Copyright (2014), with permission from Elsevier. Please click here to view a larger version of this figure.
In most cases, fully 2′-O-methylated, 5′- and 3′-phosphorylated heptamer-type sgRNAs dissolved in water-for-injection-grade water (in the concentration of 100 µM) appear to be stable at below -20 °C for at least one year, judging from their activity to induce apoptosis in cancer cells. However, their stability would change depending on their sequence, quality, and purity. In some cases, 5′- or 3′-phosphate of a part of sgRNA molecules appears to be removed spontaneously during storage, judging from mass spectrometry analysis, although this may not affect their activity seriously.
When a hit sgRNA is obtained by the library screening, its cellular target RNAs can be found by transcriptome analysis through a DNA microarray and/or next-generation sequencing. The data obtained so far, however, suggest that downregulation levels of target RNAs via heptamer-type sgRNAs are usually moderate regardless of the presence or absence of transfection reagents10,12,14,17,18.
The advantage of heptamer-type sgRNAs targeting BCL2 and WT1 mRNAs is that they can efficiently induce apoptosis in human leukemia cells in spite of weak downregulation of the target mRNAs17,18, suggesting the presence of additional RNA targets and/or additional mechanisms other than TRUE gene silencing.
Since even the screening of the library containing only 156 heptamer-type sgRNAs can make 20 hits, around 2,000 hits would be expected to be obtained by screening the full-scale library composed of 16,384 heptamers22,25.
For future applications, sgRNAs other than the heptamer type can be more suitable for TRUE gene silencing11–13,15,16, although heptamer-type sgRNAs are easier, more accurate and cheaper to synthesize than longer sgRNAs. Once the therapeutic sgRNA candidate is obtained, it is easy to proceed to animal model experiments.
The authors have nothing to disclose.
This work was supported by Adaptable and Seamless Technology Transfer Program through Target-driven R&D, Japan Science and Technology Agency, the Science Research Promotion Fund from the Promotion and Mutual Aid Corporation for Private Schools of Japan, and the JSPS KAKENHI Grant Numbers 24300342 and 15H04313.
Custom heptamer RNA | Nippon Bioservice | ||
OligoSep Prep HC Cartridge | Transgenomic | 99-3860 | |
Water-for-injection-grade Water | Otsuka Pharmaceutical | 7131400A2129 | |
RPMI-1640 medium | Wako | 189-02025 | |
Fetal Bovine Serum | SIGMA-ALDRICH | 172012-500ML | |
Penicillin-Streptomycin Mixed Solution | Nacalai Tesque | 26253-84 | |
96 Well Plate | Greiner Bio-one | 655 180 | |
Cell Counting Kit-8 | DOJINDO | 343-07623 | WST-8 solution |
Microplate Reader, Sunrise Thermo RC-R | TEKAN | 510-82851 | |
FACS Round-Bottom Tube | BD Falcon | 60819-820 | |
Phosphate Buffered Saline | SIGMA-ALDRICH | P7059-1L | |
PE Annexin V Binding Buffer | BD Biosciences | 51-66121E | |
PE Annexin V | BD Biosciences | 51-65875X | |
7AAD | SIGMA-ALDRICH | A9400-1MG | |
Flow Cytometer, FACSCalibur | BD Biosciences | 342973 |