TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents

Summary

Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers.

Abstract

TRUE gene silencing (termed after tRNase Z^L-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 Z^L, 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.

Introduction

TRUE gene silencing (termed after tRNase Z^L-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies¹. This technology has been developed based on properties of tRNase Z^L (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 sgRNA^2-9. sgRNA, which is usually 7-31 nt in length, is categorized into four groups, 5′-half-tRNA....

Protocol

1. Construction of a Heptamer-type sgRNA Library

1. Select a subset of the 16,384 (4⁷) 7-nt sequences unless the full-scale library is to be constructed.
   NOTE: The sequences can be random and/or designed to target specific cellular RNAs. For the latter, find hairpin structures resembling the T-arm of tRNA in a target RNA with the aid of an appropriate computer program and/or visually, and select sequences complementary to 7-nt sequences immediately downstream of the hairpin structures^4,10,17,18.
2. Synthesize each of the selected heptamers as a fully 2′-O-methylated, 5′- and 3′-phosphorylated RNA ....

Results

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 cel.......

Discussion

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 du.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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