Fast mutagenesis system

The wild-type flaviviral xrRNAs and their mutant constructs were generated as follows. The plasmid encoding an upstream T7 promoter and a flaviviral xrRNA sequence was synthesized and sequenced by Wuxi Qinglan Biotechnology Inc, Wuxi, China. All the mutants were generated using Transgen’s Fast Mutagenesis System. The double-stranded DNA fragment templates for in vitro RNA production were generated by PCR using a common upstream forward primer targeted the plasmids and a downstream reverse primer specific to the respective plasmids. The RNAs were in vitro transcribed using T7 RNA polymerase and purified by preparative, non-denaturing polyacrylamide gel electrophoresis, the target RNA bands were cut and passively eluted from gel slices into buffer containing 0.3 M NaOAc and 1 mM EDTA, pH 5.2 overnight at 4 °C. The RNAs were further passed through the size-exclusion chromatography column to the final buffer condition for SAXS and Xrn1 resistance experiments. The sequences for all the constructs and the primers used in this study are listed in Supplementary Tables S1 and S2, respectively.

Bioinformatic tools (PicTar and TargetScan algorithms) were used to predict the targets of miR‐29b‐3p. The NOTCH2 3′ UTR from human/mouse/rat genomic DNA was cloned into XhoI and NotI sites downstream of Renilla luciferase in the psiCHECK‐2 vector (Promega), while the firefly luciferase gene was used as an internal control. Mutation of the NOTCH2 3′ UTR was performed using Fast Mutagenesis System (TransGen Biotech). The PCR primer is listed in Table S5. For analysis of luciferase activity, HEK293, HL1 and H9c2 cells were cultured in 96‐well plates and co‐transfected with 100 ng of psiCHECK‐2 vector containing the 3′ UTR of NOTCH2 (WT or MUT) and 20 pmol of miRNA mimic per well. The four groups were psiCHECK‐2‐NOTCH2‐WT+ miR‐NC mimic, psiCHECK‐2‐NOTCH2‐WT+ miR‐29b‐3p mimic, psiCHECK‐2‐NOTCH2‐MUT+ miR‐NC mimic and psiCHECK‐2‐NOTCH2‐MUT+ miR‐29b‐3p mimic. The luciferase analysis was performed 24 hours later with the Dual‐Luciferase Reporter Assay (Promega). After lysed for 15 minutes at room temperature, the relative luciferase activity was obtained after normalization to firefly activity.

To perform the proof-of-concept experiment of the mismatch-tolerant RT-LAMP, a series of mutants that form mismatches with the F3, FIP and BLP primers were constructed using a fast mutagenesis system (TransGen, Beijing, China) based on the 3′-UTR region of the DENV-4 genome. In brief, the 3′-UTR fragment of DENV-4 was obtained using StarScript II Probe One-Step qRT-PCR Kit (GenStar, Beijing, China) with primers F3/134 and B3/4, and then sub-cloned into pMD18-T plasmid vector (TaKaRa, Dalian, China). A T7 promoter was fused to the 5′-end of the primer F3/134. Various mutants were constructed using site-directed mutagenesis with mutagenic primers (Supplementary Table S1) and confirmed by Sanger sequencing. Mutant RNAs were obtained through in vitro transcription with T7 RNA polymerase, and quantified using spectrophotometric absorbance at 260 nm (NanoDrop, Technologies Inc.). Copy number of each mutant RNA was calculated using the following formula: RNA copies/μl = [RNA concentration (g/μl)/(nt transcript length × 340)] × 6.022 × 10²³.

Site-directed mutagenesis was performed using Fast Mutagenesis System (Trans Gen Biotech) following the manufacturer’s instructions. The plasmids pMV261- msmeg_1954 and pGEX-6p-1-msmeg_1954 were used as the corresponding template for constructing a series of mutants for overexpression in M. smegmatis and for protein purification in E. coli, respectively. The pMV261-msmeg_1954 mutants were transformed into M. smegmatis mc²155, and pGEX-6P-1-msmeg_1954 mutants were transformed into E. coli BL21-Codon Plus (DE3) RIL. The N-terminal truncation of 98 residues (MSMEG_1954N^△98) was constructed into pMV261 and transformed into M. smegmatis mc²155 to obtain pMV261- msmeg_1954N^Δ98-mc²155.

The fragment from Bcl2-3′-UTR containing the predicted miR-125b-5p binding site was amplified by PCR and then cloned into a pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega) to form the reporter vector Bcl2-3′-UTR wild type. The putative binding site of miR-125b-5p in the Bcl2- 3′-UTR was mutated by using a site-directed mutagenesis kit from Fast Mutagenesis System (TransGen Biotech, Beijing, China), and the mutant reporter vector was named as Bcl2-3′-UTR mutant. The miR-125b-5p mimic and vector were co-transfected into 3 human gallbladder cancer cells, and Renilla luciferase reporter plasmid (pRL-TK) was also co-transfected as the internal reference. After transfection for 48 h, cells were lysed in passive lysing buffer, and then firefly and Renilla luciferase activities were analyzed using the Dual-Luciferase Reporter Assay System (Promega). The results of firefly luciferase activity were normalized to the Renilla luciferase activity.