We recently developed a method for generating functional-RNA arrays and demonstrated that they could be used to detect fluorogen-RNA aptamer interactions. Specifically, we were able to visualise the interaction between malachite green and the malachite green-binding aptamer. Here we expand this study to demonstrate that functional-RNA arrays can be used to quantify fluorogen-aptamer interactions. As proof-of-concept, we provide detailed protocols for the production of malachite green-binding RNA aptamer and DFHBI-binding Spinach RNA aptamer arrays. Furthermore, we discuss the potential utility of the technology to fluorogen-binding RNA aptamers, including application as a molecular biosensor platform. We anticipate that functional-RNA array technology will be beneficial for a wide variety of biological disciplines.The discovery and engineering of more and more functions of RNA has highlighted the utility of RNA-targeting small molecules. Recently, several fluorogen-binding RNA aptamers have been developed that have been applied to live cell imaging of RNA and metabolites as RNA tags or biosensors, respectively. Although the design and application of these fluorogen-binding RNA aptamer-based devices is straightforward in theory, in practice, careful optimisation is required. For this reason, high throughput  The earliest examples of RNA-targeting small molecules were the antibacterial translation inhibitors that have been used both therapeutically and as functional probes  Once synthesised, the small molecule-binding RNA must bind to an RNA capture surface to generate the RNA array. We decided to use immobilisation RNA aptamers encoded 3′ to the small molecule-binding RNA to facilitate the tethering of the small molecule-binding RNA to a capture surface coated with the immobilisation RNA aptamer’s cognate ligand. The use of a structured RNA aptamer in the 3′ location serves as an internal control for RNA folding and ensures that only fully-transcribed small molecule-binding RNAs are immobilised. Critical considerations when selecting an immobilisation RNA aptamer include the affinity for the cognate ligand; the availability of the cognate ligand; and the availability of surface-immobilisation strategies for the cognate ligand. We have demonstrated proof-of-concept with the tobramycin-binding RNA aptamer in vitro transcription templates to evaluate the potential effects of the linker sequence on small molecule-binding RNA and streptavidin-binding RNA aptamer folding.Since function of the small molecule-binding RNA and the streptavidin-binding RNA aptamer will depend on them adopting their correct structures, an unstructured linker is included between the small molecule-binding RNA and the streptavidin-binding RNA aptamer to promote the independent folding of the two RNAs. A variety of linker sequences have been used successfully and these are presented in in vitro transcription templates on the array. We use a 5′ fluorescently labelled reverse primer complementary to the 3′ end of the sequence encoding the streptavidin-binding RNA aptamer to incorporate the fluorescent tag during template synthesis. A variety of fluorophores can be incorporated in this manner and choice of fluorophore will be dependent on the detection capabilities of the microarray slide scanner to be used. We typically use Dy-549.Finally, the optional 3′ fluorescent tag allows for visualisation and quantification of the DNA 2.3in vitro transcription templates must conform to the overall design principles discussed in methods and approaches in vitro transcription templates used in this manuscript.The 5′ biotinylated, or 5′ biotinylated and 3′ Dy-549-labelled, double-stranded DNA 2.3.1DNA oligonucleotide primers .dNTPs .Macherey-Nagel NucleoSpin Gel and PCR Clean-up Kit .2.3.2GeneAmp PCR System 9700 thermocycler (Applied Biosystems).2.3.3https://hpcwebapps.cit.nih.gov/dnaworks/) which, by default, designs primers based on thermodynamically balanced (TB) gene synthesis. Variable parameters are set to a Tm of 62 °C and oligo lengths between 60 and 80 nucleotides. If TB gene synthesis is unsuccessful, primers can be redesigned in DNAWorks using TBIO  mode. The primers that were used for gene synthesis in this manuscript are presented in Non-labelled PCR templates are initially generated by gene synthesis 2.4in vitro transcription template array and the small molecule-binding RNA array require a streptavidin-coated capture surface. It is important to be aware that the use of streptavidin-coated capture surfaces for both the DNA in vitro transcription templates and the small molecule-binding RNAs could result in the in vitro transcribed RNA binding to the DNA capture surface, rather than to the RNA capture surface, as intended. We minimise any potential problems associated with this by controlling the density of streptavidin on the capture surfaces. Therefore, although streptavidin-coated microarray slides are commercially available, we prepare them using NHS-activated microarray slides, commercially available purified streptavidin and standard amine coupling chemistry. This approach allows us to prepare an RNA capture surface with a higher density of streptavidin than the DNA capture surface. Alternative solutions include using orthogonal capture-systems for the DNA and RNA, or the DNA capture surface could be blocked with biotin following DNA capture.Both the production of the DNA 2.4.1NEXTERION Slide H NHS-activated microarray slides LifterSlips .Streptavidin .Phosphate-buffered saline (PBS) .Tween 20 .Ethanolamine .2.4.2Humidified chamber comprising a Labnet Mini incubator , containing a glass bowl with 25 ml water.Denley Spiramix rolling platform (Thermo Scientific).2.4.32O for 5 min, with rolling, at room temperature. The streptavidin-coated capture surface is then incubated in 45 ml 50 mM ethanolamine-HCl pH 8.5 for 30 min, with rolling, at room temperature, to block any unreacted NHS functional groups. A second wash cycle is performed: (i) 45 ml PBST for 5 min, with rolling, at room temperature (ii) 45 ml PBS for 5 min, with rolling, at room temperature and (iii) 45 ml H2O for 5 min, with rolling, at room temperature. The streptavidin-coated capture surface is dried by centrifugation at 500 × g for 5 min at room temperature. The streptavidin-coated capture surfaces can be stored at −20 °C for at least two weeks, until required.90 µl of 1 µM streptavidin (DNA capture surface) or 16.7 µM streptavidin (RNA capture surface) in PBS, pH 7.4, is pipetted onto a NHS-activated microarray slide . This is2.5in vitro transcription templates are prepared by immobilising 5′ biotinylated in vitro transcription templates onto streptavidin-coated DNA capture surfaces in an array format. The biotinylated templates can be spotted onto the streptavidin-coated DNA capture surface manually with a pipette  or with an automated arrayer . In our experience, similar results are obtained from both manual and automated spotting in vitro transcription template within a spot rather than on the absolute amount of DNA in vitro transcription template present. Reducing the spotting volume, by manually pipetting a smaller volume or using an automated arrayer, also reduces the spot size which offsets any effect on DNA density. The primary advantage of automated spotting is not increased sensitivity, rather, it is the smaller spot size that enables increased spot density and increased throughput. Here we focus on high-spot density arrays that are produced using an automated arrayer. However, the principles described are equally applicable to low- to medium-spot density arrays produced by manual spotting. In the first instance, it is advisable to spot each DNA in vitro transcription template at a range of concentrations because variability in transcription efficiency and probe sensitivity can affect the limits of detection. We typically start by spotting DNA in vitro transcription template prepared at concentrations ranging from low nM to low μM.Template arrays of DNA 2.5.1in vitro transcription templates .Tween 20 .2.5.2Qarray 2 arrayer (Genetix).Denley Spiramix rolling platform (Thermo Scientific).2.5.3in vitro DNA template, in PBS, pH 7.4, onto a streptavidin-coated DNA capture surface (in vitro transcription template array is briefly dipped in PBST and then washed with: (i) 45 ml PBST for 5 min, with rolling, at room temperature (ii) 45 ml PBS for 5 min, with rolling, at room temperature and (iii) 45 ml H2O for 5 min, with rolling, at room temperature. It is dried by centrifugation at 500 × g for 5 min at room temperature. The DNA in vitro transcription template array can be stored at −20 °C for at least two weeks, until required.An automated arrayer is used to spot a concentration range of 5′ biotinylated, or 5′ biotinylated and 3′ fluorescently labelled  surface . This is2.6http://imagej.nih.gov/ij/, 1997–2016).We have access to a microarray scanner system that includes both the microarray slide scanner and integrated image analysis software. However, densitometry can also be performed using stand-alone image analysis software such as ImageJ .2.6.2in vitro transcription template arrays are visualised with a microarray scanner using an excitation wavelength of 532 nm and a Standard Green filter. The fluorescence intensity of each spot on the array is quantified using the integrated image analysis software on the microarray scanner.Dy-549 fluorescently-labelled DNA 2.7in vitro transcription template array – in vitro transcription reagent solution – streptavidin-coated RNA capture surface .MEGAscript T7 Transcription Kit .PBS .Tween 20 .2.7.2Humidified chamber comprising a Labnet Mini incubator , containing a glass bowl with 25 ml water.2.7.3in vitro transcription template array – in vitro transcription reagent solution – streptavidin-coated RNA capture surface “sandwich” is assembled as shown in in vitro transcription reagent solution  is pipetted onto the RNA capture surface. A DNA in vitro transcription template array is positioned on top of the RNA capture surface, array side down. This assembly is incubated in a humidified environment at 37 °C for 90 min .MEGAscript T7 Transcription Kit .Probe (see results and applications section for details).PBS .Tween 20 .2.8.1.2Humidified chamber comprising a Labnet Mini incubator , containing a glass bowl with 25 ml water.GenePix 4300A microarray scanner with integrated GenePix Pro 7 image analysis software (Molecular Devices).2.8.1.3in vitro transcription template array – in vitro transcription reagent solution – streptavidin-coated RNA capture surface “sandwich” is assembled as shown in in vitro transcription reagent solution  supplemented with the appropriate small molecule probe(s) (see results and applications section for details) is pipetted onto the RNA capture surface. A DNA in vitro transcription template array is positioned on top of the RNA capture surface, array side down. This assembly is incubated in a humidified environment at 37 °C for 90 min. The sandwich is disassembled and the newly generated, probed small molecule-binding RNA array is placed in a 50 ml Falcon tube and washed with: (i) 45 ml PBST for 5 min, with rolling, at room temperature (ii) 45 ml PBS for 5 min, with rolling, at room temperature and (iii) 45 ml H2O for 5 min, with rolling, at room temperature. It is then dried by centrifugation at 500 × g for 5 min at room temperature. The probed small molecule-binding RNA array is visualised using the appropriate excitation wavelength and emission filter for the fluorogen-binding RNA system (see results and applications section for details) using a microarray slide scanner. The fluorescence intensity of each spot on the array is quantified using the integrated image analysis software on the microarray scanner.The DNA 2.8.22.8.2.1LifterSlips of 24 mm × 60 mm .Probe (see results and applications section for details).Probe buffer (see results and applications section for details).2.8.2.2Humidified chamber comprising a Labnet Mini incubator , containing a glass bowl with 25 ml water.GenePix 4300A microarray scanner with integrated GenePix Pro 7 image analysis software (Molecular Devices).2.8.2.390 µl of small molecule probe in probe buffer (see results and applications section for details) is pipetted onto the small molecule-binding RNA array and covered with a LifterSlip . This is33.13.1.1in vitro transcription template arrays of Dy-549 fluorescently-labelled or non-labelled templates, encoding the malachite green-binding aptamer (MGSA), were prepared , 20 mM NaCl, 6 mM MgCl2 and probe for 2 to 16 h at room temperature. The RNA array is then placed in a foil-covered 50 ml Falcon tube and is washed with: (i) 45 ml H2O for 1 min, with rolling, at room temperature; (ii) 45 ml of H2O for 10 s, with rolling, at room temperature. This is followed by a final dip in 45 ml H2O at room temperature before drying by centrifugation at 500 × g for 5 min at room temperature. Probing of a MGSA RNA array with 40 µM malachite green  for 2 h at room temperature post RNA array production resulted in detectable fluorescence from the malachite green-binding RNA aptamer–malachite green complex using an excitation wavelength of 635 nm and a Standard Red filter  in 2X SSC, 0.1% (w/v) SDS for 30 min at room temperature. The probed MGSA RNA array was placed in a foil-covered 50 ml Falcon tube and was washed with: (i) 45 ml PBST for 5 min, with rolling, at room temperature; (ii) 45 ml H2O for 30 s, with rolling, at room temperature. This was followed by a final dip in 45 ml H2O at room temperature before drying by centrifugation at 500 × g for 5 min at room temperature. Detectable fluorescence was observed using an excitation wavelength of 635 nm and a Standard Red filter , were prepared  and visualising using an excitation wavelength of 488 nm and a Standard Blue filter  and visualised as described for the MGSA aptamer in in vitro transcription template concentrations between 22 and 330 nM , were prepared  and a Standard Blue filter   D.Fig. 9ASA RNA array was washed with: (i) an initial dip in 40 mM Tris-HCl, pH 7.8, 20 mM NaCl, 6 mM MgCl2; (ii) 45 ml 40 mM Tris-HCl, pH 7.8, 20 mM NaCl, 6 mM MgCl2 for 1 min, with rolling, at room temperature. This was followed by a final dip in 45 ml H2O at room temperature before drying by centrifugation at 500 × g for 5 min at room temperature.To confirm the RNA levels, the Spinach/SAMSA RNA array was placed in a foil-covered 50 ml Falcon tube and was washed with: (i) an initial dip in 40 mM Tris-HCl, pH 7.8, 20 mM NaCl, 6 mM MgCl2; (ii) 45 ml 40 mM Tris-HCl, pH 7.8, 20 mM NaCl, 6 mM MgCl2 for 1 min, with rolling, at room temperature. This was followed by a final dip in 45 ml H2O at room temperature before drying by centrifugation at 500 × g for 5 min at room temperature. Detectable fluorescence was observed using an excitation wavelength of 635 nm and a Standard Red filter  in 2X SSC, 0.1% (w/v) SDS for 30 min at room temperature. The probed Spinach/SAMd filter E and was3.2.13.2.1.1in vitro applications in mind (e.g. iSpinach A number of Spinach-based non-fluorescent/fluorogenic small molecule biosensors have been engineered. However, their design required optimisation of the connecting region between the Spinach aptamer and the non-fluorescent/fluorogenic small molecule-binding aptamer (reviewed in 3.2.1.2As more and more fluorogen-binding RNA aptamer-based biosensors are developed, this opens up the possibility of using RNA arrays as a screening platform for biomarkers. Several diseases have known biomarker fingerprints e.g. cancers 4in vitro transcription templates to be individually designed and synthesised currently limits the throughput capabilities of RNA arrays. However, computer programs could be developed to aid the design of DNA in vitro transcription templates and robotics could be employed for the DNA synthesis step. Automation of these, and possibly subsequent steps, could easily allow the high-throughput potential of RNA array technology to be realised.There are clearly hurdles to overcome before RNA arrays can routinely be applied as diagnostic tools. For example, biosensors will be required that can detect a wide range of biomarkers including small molecules and also larger macromolecules such as proteins and nucleic acids. In addition, there is the challenge of stabilising RNA so that it is not degraded in the presence of biological samples. However, combining SELEX approaches with RNA array screening could guide the development of these biosensors such that the final diagnostic platform could be manufactured using available RNA stabilisation chemistries 5Here we have provided a detailed protocol for generating functional-RNA arrays for the purpose of investigating RNA-targeting small molecules. Specifically, we have demonstrated the capabilities of RNA arrays with respect to fluorogen-binding RNA aptamers and fluorogen-binding RNA aptamer-based biosensors. We have also discussed the potential applications of RNA arrays to the design, optimisation and utility of fluorogen-binding RNA aptamers. We anticipate that, in these capacities, RNA arrays will prove to be a valuable tool for a range of disciplines including molecular biology, metabolic engineering, synthetic biology and medicine.


NSUN2 is an RNA methyltransferase responsible for the m5C modification of multiple RNAs. In this study, we knocked down the NSUN2 gene in HepG2 cells by CRISPR/Cas9 technology and performed high-throughput RNA-BisSeq. An important tumor-related lncRNA H19 was identified to be targeted by NSUN2. Studies have shown that the expression of H19 lncRNA is abnormally elevated and has a carcinogenic effect in many types of tumors. Our results demonstrated that m5C modification of H19 lncRNA can increase its stability. Interestingly, m5C-modified H19 lncRNA can be specifically bound by G3BP1, a well-known oncoprotein which further leads to MYC accumulation. This may be a novel mechanism by which lncRNA H19 exerts its oncogenic effect. Besides, both the m5C methylation level and the expression level of H19 lncRNA in hepatocellular carcinoma tissues were significantly higher than those in adjacent non-cancer tissues, which were closely associated with poor differentiation of hepatocellular carcinoma (HCC). In conclusion, we found that H19 RNA is a specific target for the NSUN2 modifier. The m5C-modified H19 lncRNA may promote the occurrence and development of tumors by recruiting the G3BP1 oncoprotein. Our findings may provide a potential target and biomarker for the diagnosis and treatment of HCC.RNA methylation is an important epigenetic modification. Recent studies on RNA methylation mainly focus on the m The function of DNA m5C modification in carcinogenesis has been studied extensively. However, very little is known about the function of RNA m5C modification in tumorigenesis. NSUN2, also named Misu [5C-modifying methyltransferases in mammals and plays an important role in RNA metabolic processes, such as RNA processing [In recent years, the association between abnormal RNA modifications and tumorigenesis has attracted widespread attention. Several studies have clarified that N6-methyladenosine  in a variety of RNA species, including tRNA, rRNA, mRNA, lncRNA, and mitochondrial RNAs [5C modifications are detected in lncRNAs [NSUN2 was shown to account for the formation of 5-methylcytosine  was employed to detect NSUN2 deficiency induced genome-wide expression alterations. In total, 2681 differentially expressed genes (DEGs) were found between NSUN2-deficient HepG2 and wild-type HepG2 cells Fig. .Fig. 3Ov5C candidate sites in wild-type cells, while the number of m5C candidate sites in NSUN2-deficient HepG2 cells was considerably reduced to only 3182 m5C candidate sites  analysis was performed to map and compare transcriptome 5-methylcytosine sites between NSUN2-deficient HepG2 cells and wild-type HepG2 cells  screened by RNA-BisSeq were further subjected to GO and KEGG pathway enrichment analyses to identify the biological role of NSUN2-mediated RNA m5C methylation. The significantly enriched GO terms of DMGs between NSUN2-deficient HepG2 and wild-type cells were associated with translational initiation , membrane (ontology: cellular component) and poly(A) RNA binding (ontology: molecular function). KEGG pathway analysis show enrichment of these DMGs in 20 pathways, including spliceosome, proteoglycans in cancer, cell cycle, estrogen signaling pathway, etc.  were randomly selected for subsequent bisulfite-PCR amplification and Sanger sequencing. The results indicated that all of the Cs were converted to Ts except the sites identified by RNA-BisSeq  treatment followed by RT-qPCR to analyze the H19 RNA half-life. As shown in Fig. H19 RNA was markedly shortened after NSUN2 depletion. Furthermore, NSUN2 re-expression restored the half-life of H19 RNA to a certain extent in NSUN2-deficient HepG2 cells  or fragment with a mutated m5C site (H19-Mut). As expected, mutation of m5C site significantly inhibited luciferase activity in normal HepG2 cells but not NSUN2-deficient HepG2 cells  with its RNA methylation level in these tissues  associated with the differentiation stages of tumors  were identified between the RNA m5C modification of H19 and other clinicopathological characteristics, including patient age, sex, extrahepatic metastasis, liver cirrhosis, and tumor size  using H19-specific probes and subsequent mass spectrometry analysis  assay using a G3BP1 antibody  was confirmed to bind methylated H19 RNA based on the presence of NSUN2. Moreover, lncRNA H19 was shown to be overexpressed, and its overexpression accompanied by hypermethylation in HCC tissues compared to matched normal liver tissues; in addition, high levels of H19 RNA methylation and expression were correlated with HCC poor differentiation. It is well known that tumor poor differentiation is an important feature of malignant tumors, which directly affects tumor progression and prognosis. Taken together, these results suggest that abnormal NSUN2-mediated RNA m5C modification represents a mechanism for the progression of human maglinant HCC, and it may serve as a prognostic biomarker to distinguish HCC patients.Dysregulation of epigenomic modifications, including DNA methylation, histone modifications, and RNA methylation, plays an important role in tumorigenesis. In recent years, accumulating evidence has demonstrated that RNA m cancers –29. NSUNCDK1 mRNA regulates CDK1 translation and thus affects the cell cycle [Interestingly, NSUN2 is a direct target gene of MYC and is upregulated upon MYC activation. RNAi of NSUN2 blocked MYC-induced keratinocyte proliferation and cell-cycle progression. The growth of human squamous cell carcinoma xenografts was also inhibited by NSUN2 knockdown . Here, wll cycle . FurtherTranscriptome sequencing identified 2681 DEGs and 1208 DMGs in HepG2 cells. Among them, 52 genes including the lncRNA H19 had changes in both RNA expression and methylation level. In many recent studies, H19 was identified as an onco-lncRNA and to promote tumorigenesis, such as in glioblastoma , colorec5C modification may exert its function mainly by affecting translation of the RNA rather than by altering the RNA level [5C modification stabilizes oncogene RNA through the “reader” YBX1, and hypermethylated m5C levels correlate with oncogene mRNA overexpression in UBC cells [H19 RNA, which might be regulated by NSUN2-mediated m5C modification, since mutation of the methylation site could significantly inhibit luciferase activity of reporter gene in normal HepG2 cells but not NSUN2-deficient HepG2 cells.Depletion of NSUN2 lead to reduced methylation of H19 RNA, which was also accompanied by decreased expression of H19. Many previous reports have shown that NSUN2-mediated RNA mNA level , 11. A rBC cells . NSUN2 mH19 lncRNA has been confirmed to be highly expressed in many organs from early embryonic development to the fetal stage, but not after birth [H19 RNA is observed in HCC tissues compared to matched normal liver tissues, which is also correlated with HCC malignancy. Unexpectedly, the expression and methylation level of H19 RNA did not show significant correlation with clinical indicators such as tumor size and metastasis. It seems that H19 may not be the best target for the function of NSUN2 in tumor which NSUN2 deficiency significantly inhibited cancer cell proliferation and migration. We speculate that this may be due to improper sample selection. For example, in 55 clinical cases, 41 cases did not show metastasis, while only 14 cases were metastatic. In addition, the limited sample size may also be one of the reasons for this phenomenon. Moreover, NSUN2 may participate in regulating tumor progression through multiple mechanisms, which needs more research to explore. Maybe, further study in more tumor samples and multiple types of tumors can solve this. Abnormally high level of lncRNA and DNA m5C hypermethylation have been used as diagnostic tools in many types of tumors. Our study provided a mechanism by which RNA m5C methylation may act as a new diagnostic marker for tumors.er birth . Howeverer birth , lung caer birth , and blaer birth . SimilarH19 RNA to bind with other proteins. Through ChIRP assays, we found that G3BP1 specifically binds to H19 RNA in normal HepG2 cells but not in NSUN2-deficient HepG2 cells, and RIP assays also confirmed this specific interaction. Ras-GTPase-activating protein SH3 domain-binding protein 1 (G3BP1), a known oncoprotein, is overexpressed in a variety of human cancers, such as breast cancer [6A and to positively regulate mRNA stability in an m6A-regulated manner [5C [H19 RNA by recognizing m5C, and this was confirmed by further rEMSA assay. G3BP1 specific binds m5C modified H19 oligos, but not oligos without m5C. These results therefore suggested that NSUN2-mediated methylation of H19 RNA regulated its interaction with G3BP1 protein.Most lncRNAs function by binding to proteins. A previous study demonstrated that methylated cytosine of the lncRNAs XIST and HOTAIR affects their recruitment of the chromatin-modifying complex PRC2 . We testt cancer , renal ct cancer , and hept cancer . G3BP1 wd manner . BesidesH19 RNA to G3BP1 and then affect the progression and malignancy of cancer cells. Moreover, G3BP1 was shown to bind MYC mRNA and promote its decay [H19 RNA may compete with MYC mRNA for binding to G3BP1, promoting tumor cell progression. Hypomethylated H19 RNA will lose binding ability of G3BP1, and lead to an increase in binding ability between G3BP1 and MYC mRNA, which increases the decay rate and decreases the mRNA level of MYC, thus suppressing cell proliferation. In addition, MYC has been shown to bind to the promoter region of H19 and promote the transcription of H19 RNA [H19 RNA level. Thus, we propose a regulatory axis: MYC-NSUN2-H19-G3BP1-MYC . For the details of cell culture, see the Supplementary of All primers used for plasmid construction are shown in Additional file The NSUN2-deficient HepG2 cell line was generated via a CRISPR-Cas9-mediated method as previously described . For theFifty-five pairs of HCC tissues and matched non-cancerous liver tissues were surgically removed following the guidelines approved by the Institutional Review Board of Chinese PLA General Hospital. For the details, see the Supplementary of The specific primers used for amplification are shown in Additional file Cell viability was determined using the CCK-8 assay. For the details, see the Supplementary of Colony formation assay was performed as previously described with several modifications . For theA wound healing assay was performed as previously described with several modifications . For theTranswell invasion assay was performed as previously described with several modifications . For theAll mice experiments were performed according to the institutional guidelines and were approved by the Institutional Animal Care and Use Committee. For the details, see the Supplementary of The impact of NSUN2 deletion on in vitro angiogenesis of HepG2 was determined by tube formation assay. For the details, see the Supplementary of Gene-expression profiling were described in previous studies . For the5C sites are listed in Additional file RNA-BisSeq were described in previous studies . PrimersPrimers for amplification and sequencing and nucleotide dispensation orders are listed in Additional file H19 RNA was assessed by the addition of actinomycin D  into the cell culture medium. For the details, see the Supplementary of The stability of the HepG2 cells and NSUN2-deficient HepG2 cells were seeded into 24-well plates 24 h before transfection with pmirGLO-derived vectors. Forty-eight hours after transfection, cell lysates were collected and the firefly and Renilla luciferase activities were measured with a Dual Luciferase Reporter Assay system (Vazyme) following the manufacturer’s instructions. All firefly luciferase measurements were normalized to Renilla luciferase measurements from the same sample.Anti-NSUN2 , anti-G3BP1 , and anti-GAPDH  antibodies were used. For the details of western blot, see the Supplementary of CHIRP was performed as previously described with slight modifications . For theThe RIP experiment was performed as described by Tenenbaum et al.  For the rEMSA was performed as previously described with some modifications . For theAll statistical results were analyzed using IBM SPSS Statistics 21 software. For the details, see the Supplementary of Supplementary Materials and MethodsAdditional file 1Additional file 2Additional file 3AAdditional file 3BAdditional file 4


The Cas13a system has great potential in RNA interference and molecular diagnostic fields. However, lacking guidelines for crRNA design hinders practical applications of the Cas13a system in RNA editing and single nucleotide polymorphism identification. This study posits that crRNAs with hairpin spacers improve the specificity of CRISPR/Cas13a system (termed hs‐CRISPR). Gibbs free energy analysis suggests that the hairpin‐spacer crRNAs (hs‐crRNAs) suppress Cas13a's affinity to off‐target RNA. A hepatitis B virus DNA genotyping platform is established to further validate the high‐specificity of hs‐CRISPR/Cas13a system. Compared to ordinary crRNA, hs‐crRNAs increase the specificity by threefold without sacrificing the sensitivity of the CRISPR/Cas13a system. Furthermore, the mechanism of the Cas13a/hs‐crRNA/target RNA composition is elucidated with theoretical simulations. This work builds on the fundamental understanding of Cas13a activation and offers significant improvements for the rational design of crRNA for the CRISPR/Cas13a system. This study proposes that hairpin‐spacer crRNAs (hs‐crRNA) improve the specificity of CRISPR/Cas13a system (hs‐CRISPR). Gibbs free energy analysis and experimental data demonstrate that hs‐crRNAs suppress Cas13a's activity to nonspecific targets. This work provides a fundamental understanding of Cas13a activation associated with binding energetics and an efficient strategy for high‐specificity crRNA design. The activity of CRISPR/Cas13a, an RNA‐guided RNase, is determined by higher eukaryotes and prokaryotes nucleotide‐binding (HEPN) domains. Cas13a can combine with CRISPR RNA (crRNA) to form the Cas13a/crRNA duplex, which is competent at recognizing target RNA with the guidance of crRNA‐spacer. Once assembled with target RNA, the Cas13a/crRNA/target RNA ternary complex will activate the HEPN domains to create a general RNase active site in the Cas13a protein. These properties impart CRISPR/Cas13a with advantages of rapid, accurate, high‐sensitivity, and programmability for molecular diagnostics. However, CRISPR/Cas systems are still challenged by high off‐target rates that affect the accuracy and stability of practical applications.Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR‐associated (Cas) protein systems offer various possibilities in molecular diagnostics2, 3 and/or crRNA design are prominent strategies that increase efficacy. The clarification of activation mechanisms via probing structures helps the development of high‐performance Cas13a proteins. The most common method to optimize crRNA is to introduce synthetic mismatch into the crRNA‐spacer, which promotes the identification ability of single nucleotide polymorphisms (SNPs) toward Cas13a proteins. However, Cas13a protein optimization remains challenging due to its sophisticated nature. Besides, the location of synthetic mismatch in crRNA is uncertain and its enhancing mechanism is unclear. In recent years, studies show that hairpin structures enhance the functions of RNA molecules in extensive applications, such as strand‐displacement amplification, RNA interference, aptamer biosensing, and RNA origami. Furthermore, RNA secondary structures increase the specificity of Cas9 and Cas12a systems in genetic engineering. However, little is known regarding the value of hairpin‐spacer structures in the CRISPR/Cas13a system while applied to molecular diagnostics and RNA interference.Optimizing Cas proteins19 andFigure Herein, we propose a hairpin‐spacer crRNA (hs‐crRNA) strategy that improves the specificity of the CRISPR/Cas13a system (hs‐CRISPR) by suppressing the binding affinity between Cas13a and off‐target RNA Figure 1A. First22.1trans‐cleavage  and TS to AS (“ΔG02‐[on]”) or TS to uTS (termed “ΔG02‐[off]”) using fluorescence spectrometer. The difference in ΔG02‐[on] and ΔG02‐[off] was determined as “ΔG”. For hs‐CRISPR, crRNA with low free energy was used and therefore high ΔG was obtained, making off‐target RNA unable to activate the nuclease activity of Cas13a.The principle of hs‐CRISPR is described in Figure e Figure . At firsTo verify the advanced discrimination ability of hs‐CRISPR on SNPs, we further compared hs‐crRNAs with ordinary crRNA (o‐crRNA) for HBV genotyping Figure . Conside2.2Figure −1 for hs‐crRNA1 to hs‐crRNA5, respectively) than o‐crRNA (−3.68 kJ mol−1).To explore the effects of hairpin crRNA‐spacers on Cas13a activity, we compared the capabilities of crRNAs with or without hairpin spacer for HBV DNA genotyping. As shown in  The Cas13a protein has natural fluorophores (tryptophan and tyrosine residues), allowing fluorescence analysis of conformational changes. First, we incubated Cas13a (GS) with different concentrations of crRNA ranging from 0 × 10−6 to 2.5 × 10−6m at 25 and 37 °C for 10 min, respectively . A modified Stern–Volmer equation was used to calculate the exact parameters of interaction  and the Stern–Volmer plots are displayed in Figures S2 and S3 of the Supporting Information, respectively. To investigate the types of interactions between Cas13a and crRNA, we further calculated the entropy (ΔS0) and enthalpy (ΔH0) of Cas13a/crRNA duplexes using Equation  and enthalpy (ΔH0) at different temperatures, the type of interactions between Cas13a protein and crRNAs or HBV RNA can be investigated using Van ’t Hoff equation lnKa=G01 value of o‐crRNA (−39.31 kJ mol−1) was lower than hs‐crRNAs  at 37 °C, demonstrating that ordinary crRNA possessed higher binding affinity toward Cas13a protein than hairpin‐spacer crRNAs. We believe that this phenomenon is caused by the RNA structure differences, since the molecular structure is one of the most critical factors to determine the affinity. It should be noted that both the structures of RNA molecules and the amounts of Cas13a's space occupied by crRNA would affect the affinity of the protein. To further investigate the effects of Cas13a's lower binding affinity on Cas13a/crRNA duplex formation, we incubated the Cas13a with o‐crRNA and hs‐crRNA1 for different time (0–30 min) and measured the fluorescence spectra of the duplex. As shown in Figure S4 of the Supporting Information, longer incubation time would not affect the fluorescence spectra of the duplex, indicating that both o‐crRNA and hs‐crRNA1 interacted with the Cas13a and formed the Cas13a/crRNA duplex spontaneously. Notably, the shapes of fluorescence spectra of the Cas13a/o‐crRNA and Cas13a/hs‐crRNA1 duplexes were different, suggesting that certain areas of Cas13a were occupied by o‐crRNA and hs‐crRNA, which may also influence the Cas13a's binding affinity to RNA.According to the thermodynamic results provided in Figure 2.3−6–1 × 10−6m)  at 25 and 37 °C are shown in Figure S5 of the Supporting Information.To investigate the interactions between Cas13a/crRNA duplex and target RNA, we further incubated Cas13a/crRNA duplex with various concentrations of on‐target or off‐target RNA (0 × 10) Figure . SimilarG02 represents the binding affinity of Cas13a/crRNA duplex to on‐target or off‐target RNA. ΔG02‐[on] means the ΔG02 value of Cas13a/crRNA interacting with on‐target RNA, whereas ΔG02‐[off] represents off‐target RNA. As shown in Figure G02‐[off] values were similar for all crRNAs including o‐crRNA and hs‐crRNAs at 37 °C . Whereas ΔG02‐[on] values of hs‐crRNAs  were lower than o‐crRNA (−39.06 kJ mol−1), suggesting that hairpin‐spacer crRNAs increased the binding affinity of Cas13a/hs‐crRNA duplex to on‐target RNA. Accordingly, we attributed the improved binding affinity to optimal conformation of Cas13a induced by hairpin‐spacer structures of hs‐crRNAs.Standard Gibbs free energy change ΔG01 and ΔG02 in Table G01 and ΔG02‐[on] values for all crRNAs (<−77.48 kJ mol−1) were lower than that of ΔG01 and ΔG02‐[off] values (> −76.95 kJ mol−1), indicating that the Cas13a protein possessed an overall high‐level affinity to on‐target RNA to be in the active state (AS). Notably, the difference of Cas13a's binding affinity to on‐target and off‐target RNA was determined as “ΔG” (ΔG = ΔG02‐[off] – ΔG02‐[on]). As shown in Table G values  compared to o‐crRNA (1.42 kJ mol−1). This observation indicated that hs‐crRNAs suppressed the Cas13a's overall affinity to off‐target RNA, making the Cas13a/hs‐crRNA/off‐target RNA complex unable to be in the AS due to the insufficient Gibbs free energy changes. Based on our thermodynamic results, we hypothesized that ΔG values may be associated with the specificity of Cas13a system. Since higher ΔG values were obtained using hs‐crRNAs compared to o‐crRNA, we believed that it was determined by the hairpin structures which may affect Cas13a's conformational changes during crRNA binding and target RNA recognition.Based on the Gibbs free energy analysis, we summarized the thermodynamic results of Δ2.4G and the specificity of Cas13a system, we applied ordinary crRNA and hairpin‐spacer crRNAs for HBV genotyping. The workflow of HBV genotyping is shown in Figure Table To investigate the relationship between Δ[on] and F.I.[off] mean the end‐point fluorescence intensity of on‐target and off‐target signals, respectively. As shown in Table G values of crRNAs were positively related to the specificity of the Cas13a protein. According to our experimental results of all hs‐crRNAs to on‐target and off‐target HBV, we propose a hairpin structure design strategy to enhance the specificity of the hs‐CRISPR/Cas13a system. By comparing the results of hs‐crRNA1 and hs‐crRNA2 with hs‐crRNA3, we concluded that the SNP‐induced mismatch should be contained in the stem structure of hs‐crRNA to efficiently enhance the Cas13a's specificity  of Cas13a/crRNA duplex is determined by Equation (6)DF=F.0 × 10−18m (100 copies mL−1)  of o‐crRNA, hs‐crRNA1, and hs‐crRNA2 were all 10) Figure –E. TheseTo validate the feasibility of the hs‐CRISPR/Cas13a system, we selected another 28‐nt sequence containing four random SNPs for HBV B‐type and C‐type as the target of the o‐crRNA‐a and hs‐crRNA‐a. As shown in Figure 2.5Figure H0 < 0 and ΔS0 > 0. The 5′ handle of hs‐crRNA localizes on the NTD surface  and hairpin‐spacer crRNAs increased the discrimination ability of Cas13a system by at least three‐fold than ordinary crRNA. The whole assay takes about 1 h and can be encapsulated in a single pellet which is time‐saving and easy to operate. Third, we performed theoretical simulations to illustrate the mechanisms of Cas13a nuclease activity by interacting with hairpin‐spacer crRNA and target RNA. Our findings suggest that hairpin‐spacer crRNAs are compatible with traditional Cas13a system.In this study, we presented a strategy termed hs‐CRISPR to increase the specificity of CRISPR/Cas13a system. Three main enhancements were developed and achieved. First, hairpin‐spacer crRNAs with engineered secondary structures were proposed to suppress the nonspecific activity of CRISPR/Cas13a system. CRISPR/Cas technology is reported to show high off‐target effects which impedes its applications and there lacks a rational guideline for high‐specificity Cas13a–crRNA design. Herein, we developed crRNAs with hairpin‐spacer and evaluated the binding energetics of Cas13a protein and RNA by fluorescence quenching mechanism. Our enhanced system showed a high on‐target affinity of Cas13a while suppressed the off‐target activity compared to ordinary crRNA. Second, we established an HBV genotyping platform to evaluate the feasibility of hs‐CRISPR to promote the specificity of CRISPR/Cas13a system. Our platform possessed a high sensitivity of 1 × 10In summary, this enhanced hs‐CRISPR system was verified to increase the specificity of CRISPR/Cas13a system both theoretically and experimentally in this study. By comparison of ordinary crRNAs and hairpin‐spacer crRNAs, the off‐target nuclease activity of Cas13a could be suppressed by introducing an extra secondary structure to decrease the binding affinity to off‐target RNA. Furthermore, genotyping platforms based on the enhanced hs‐CRISPR system possessed several advantages such as fast, easy‐operation and programmability. Collectively, our enhanced hs‐CRISPR system will facilitate future point‐of‐care molecular diagnostics and RNA interference based on CRSIPR/Cas13a technology.4All DNA templates and primers were synthesized from Sangon Biotechnology Co., Ltd. . The fluorophore quencher‐labeled RNA reporter was purchased from Takara Biotechnology Co., Ltd. . For nucleic acid targets, plasmids containing HBV genome of genotype B and C were provided by Prof. Y. M. Wen . All sequences and plasmids used in this study were provided in Table S1 of the Supporting Information. The LwaCas13a protein was expressed and obtained from GenScript Co., Ltd. . RPA was performed following instruction of TwistAmp Basic kit from TwistDx Inc. . The dsDNAs were purified using MinElute gel extraction kit from Qiagen . crRNAs were purified using RNAXP clean beads from Beckman Coulter . The HiScribe T7 Quick High Yield RNA Synthesis kit, murine RNase inhibitor, T7 RNA polymerase, and NTP mix buffer were purchased from New England Biolabs . MEGAclear Transcription Clean‐up kit and RNase‐free water were ordered from Thermo Fisher .The real‐time fluorescence intensity of nuclease assay was determined by a microplate reader (BioTek Synergy H1). Electrophoresis was carried out using Tanon EPS‐100 electrophoresis system. Fluorescence of Cas13a protein was measured using Agilent Technologies Cary Eclipse Fluorescence Spectrometer.−3m Tris‐HCl (pH 7.5), 10 × 10−3m EDTA, 1 m NaCl. In vitro transcription of crRNA was performed using the HiScribe T7 Quick High Yield RNA Synthesis kit overnight at 37 °C. Transcription reactions contained NTP buffer mix , T7 RNA polymerase mix, template DNA (2 µg), murine RNase inhibitor . After the synthesis of crRNA, adequate RNase‐free water, and DNase I  were added into the system to digest the DNA template by incubating at 37 °C for 15 min. The transcribed crRNAs were then purified with twofold volume of RNAXP clean beads and subsequently stored at −20 °C. The obtained crRNAs were measured by Nanodrop and analyzed by denaturing urea polyacrylamide gel electrophoresis (urea/PAGE).The designed crRNA with T7 promoter sequence was used for DNA template and annealed with a short T7 primer (5′‐TAATACGACTCACTATAGGG‐3′) in 1× annealing buffer containing 100 × 10First, HBV plasmids were amplified by RPA using TwistAmp Basic kit. RPA primers for HBV targets were designed using NCBI Primer – Blast. Notably, the HBV RPA forward primers should contain a short T7 sequence at its 5′‐end for later transcription. Second, the amplified products were evaluated by agarose gel electrophoresis and then purified by MinElute PCR purification kit . Third, the purified HBV DNA amplicons contained extended T7 sequences were transcribed into RNA using the HiScribe T7 Quick High Yield RNA Synthesis kit overnight at 37 °C. Finally, the transcribed HBV RNA were purified by the MEGAclear Transcription Clean‐up kit and then evaluated by urea/PAGE.−6m) incubated with various concentrations of crRNAs (0 × 10−6 to 2.5 × 10−6m) were measured using Agilent Technologies Cary Eclipse Fluorescence Spectrometer in cuvette. Afterward, the Cas13a/crRNA duplex was incubated with transcribed HBV RNA (concentrations ranging from 0 × 10−6 to 1 × 10−6m) and emission spectra were recorded. The excitation and emission wavelengths were set at 285 and 295–500 nm, respectively. Experiments were performed at 25 and 37 °C for all RNAs.LwaCas13a proteins have natural fluorophores (tyrosine and tryptophan residues) and allow the study of conformational changes. Intrinsic fluorescence spectra of Cas13a protein , 45 × 10−9m LwCas13a protein, 22.5 × 10−9m crRNA, 250 × 10−9m quenched fluorescent RNA reporter, 5 × 10−3m MgCl2, and 14 × 10−3m MgAc. Reactions were proceeded for 2 h at 37 °C in a microplate reader for fluorescence kinetics measurement every 2 min.The reactions of RPA‐HBV DNA amplification, T7 RNA polymerase‐in vitro transcription and Cas13a nuclease assay were integrated in one pellet. Briefly, a 25 µL of single‐pellet reaction solution consisted of 0.48 × 10http://hdock.phys.hust.edu.cn/). The 3D structure of Cas13a protein was obtained from the Protein Data Bank ID 5W1H. The 3D structures of crRNA and target RNA were generated by the RNAComposer server (http://rnacomposer.cs.put.poznan.pl/). Rigid docking calculations were carried out to investigate the binding activity between Cas13a and crRNA, followed for the Cas13a/crRNA duplex and target RNA. Besides, possible interactions were evaluated at the primary state in binary and ternary systems. The docking results were analyzed using Visual Molecular Dynamics. The positions of molecules were investigated based on the energy score. The best pose of interaction was selected based on the minimum energy HDOCK score.The molecular docking simulation was performed to predict the consensus pose corresponds to the interaction interface at the primary state in binary and ternary systems of Cas13a/crRNA and Cas13a/crRNA‐target RNA, respectively. The docking calculations were processed by the HDOCK server (edu.cn/).41 Thet‐test using Graphpad Prism 8.0 and Origin 2021. Each experiment was repeated three times. The results were presented as mean ± S.D.Statistical analysis was performed by two‐tailed Student's The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


Cap-independent translation initiation plays crucial roles in fine-tuning gene expression under global translation shutdown conditions. Translation of uncapped or de-capped transcripts can be stimulated by Cap-independent translation enhancer (CITE) elements, but the mechanisms of CITE-mediated translation initiation remain understudied. Here, we characterized a short 5ʹ-UTR RNA sequence from black beetle virus, BBV-seq. Mutational analysis indicates that the entire BBV-seq is required for efficient translation initiation, but this sequence does not operate as an IRES-type module. In yeast cell-free translation extracts, BBV-seq promoted efficient initiation on cap-free mRNA using a scanning mechanism. Moreover, BBV-seq can increase translation efficiency resulting from conventional cap-dependent translation initiation. Using genetic approaches, we found that BBV-seq exploits RNA-binding properties of eIF4G1 to promote initiation. Thus, BBV-seq constitutes a previously uncharacterized short, linear CITE that influences eIF4G1 to initiate 5′ end-dependent, cap-independent translation. These findings bring new insights into CITE-mediated translational control of gene expression. Untranslated regions at the 5ʹ end of RNA (5ʹ-UTRs), also known as leader sequences, play key roles in translation initiation, inviting intensive investigation to elucidate every aspect of their function.Translation initiation is one of the most critical regulatory steps in protein synthesis, providing control over individual mRNAs’ translational efficiency and playing an important role in stress responses6. Undoubtedly, the most important function of 5ʹ-UTRs is the recruitment of multiple translation initiation factors required for ribosomal subunit association at the correctly selected AUG start codon. Modifying the 5ʹ-end of mRNA with a 7-methyl guanosine cap structure (m7G-cap) enables it to interact with the cap-binding protein eIF4E, followed by recruitment of the scaffolding factor eIF4G and DEAD-box RNA helicase eIF4A (the eIF4F cap-binding complex)8. eIF4F-primed mRNA associates with the 43S pre-initiation complex (43S PIC) composed of a 40S ribosomal subunit bound to eIF1, eIF3, eIF2, and initiator methionyl-tRNA (tRNAiMet), forming a scanning-competent 48S initiation complex (48S IC)4. 48S IC scans mRNA until it recognizes the first AUG triplet in favorable context, and a codon-anticodon interaction is formed2, leading to weakening of ribosomal association of eIF110. The action of eIF5B evicts the initiation factors except for eIF1A during subunit joining11, and the subsequent hydrolysis of eIF5B·GTP leads to dissociation of eIF5B·GDP and eIF1A and the formation of the elongation-competent 80S complex.5ʹ-UTRs are non-coding regions within mRNAs that are located directly upstream of open reading frames (ORF). These elements vary widely in their length and structure6). For example, the Translation Initiator of Short 5ʹ-UTR (TISU) elements, that are only 12 nucleotides long, promote scanning-free initiation12. Other unstructured (linear) elements of 5ʹ-UTRs include upstream ORFs (uORFs) that lead to reduced expression of the downstream primary ORF14, while the mammalian Kozak sequence (5ʹ-GCC(A/G)CCAUGG-3ʹ) flanking the initiation codon promotes its recognition by the scanning complex to ensure fidelity of translation initiation16. Higher-order RNA structures include internal ribosome entry sites .5ʹ-UTRs not only provide the platform for cap-dependent translation initiation, but also possess multiple regulatory functions as modulators of translation initiation, ultimately contributing to the expression of a particular protein at a particular time in a particular environment . Engineered mRNA reporters carrying 5ʹ-CITEs taken from viral RNA or mammalian Apaf-1 mRNA have been shown to resist stress-induced translational repression, suggesting that like IRESs, CITEs may provide regulatory functions during unfavorable physiological conditions33. First identified within 3ʹ-UTRs of some plant viruses, CITEs constitute 7 distinct classes, each of which differs greatly in its location (5ʹ-UTR or 3ʹ-UTR), length, and secondary structure, as well as in host factors required for translation initiation (reviewed in34). A recently published study has found that CITEs exist in eukaryotic cellular mRNAs and participate in fine-tuning expression of stress-response proteins during stress conditions in a cap- and IRES-independent fashion1. However, despite their contribution to a plethora of 5ʹ-UTR-regulated initiation mechanisms, CITE elements have received only limited experimental attention.Besides IRESs, cap-independent translation initiation can be promoted through cap-independent translation enhancer elements (CITEs). Similarly to IRESs, CITEs can initiate translation without a full set of translation initiation factors38. These data suggested that this viral 5′-UTR exploits a mechanism that is widely used in different eukaryotes. BBV-seq is derived from RNA1 of BBV, a single-stranded, positive-sense RNA virus of the Nodaviridae family. For laboratory use, BBV-seq was initially described39 as an “efficient translation initiation signal” to be used under poor protein expression conditions. As predicted, BBV-seq increased yield in translation of coxsackievirus 3C protease and β-galactosidase39. Since the original statement published in 1987 (“The molecular basis of this phenomenon is not well understood”39), no published information has appeared concerning the mechanisms of BBV-seq functioning during translation. Given the growing appreciation of the variety of roles that 5ʹ-UTRs play in regulating protein synthesis, we were eager to learn more about the mechanism by which a relatively short BBV-seq influences translation in eukaryotes.In this study, we follow up on our previous work and observations by others that showed that a short 39-nucleotide sequence taken from the black beetle virus  can direct efficient translation of a downstream ORF in a wide variety of eukaryotic cells and cell extracts 38, we reasoned that the yeast-derived translational system would be an appropriate experimental platform. In addition, the role of individual protein factors in translation could be interrogated by using translation-active extracts prepared from genetically modified yeast strains. Furthermore, we verified that the efficiency of translation driven by BBV-seq in yeast lysates was comparable to that of the widely used Rabbit Reticulocyte Lysate .We began addressing the enigmatic nature of BBV-seq by examining whether this sequence is capable of promoting translation initiation in a cap-independent manner, and whether it functions as an enhancer/regulatory element in conventional cap-dependent translation. To this end, we set up translation reactions using cell-free, translationally competent yeast lysates charged with recombinant RNAs. Given that BBV-seq was previously used as a tool to increase protein expression in yeast cellsRenilla luciferase ; this dual-protein reporter was cloned into the pYes vector plasmid downstream of the T7 promoter, while various 5ʹ-UTRs were placed immediately upstream of the reporter’s ATG start codon  protein tag was fused with the gene for ays Fig. a-4. The 43. Reactions were incubated at 21 °C, consistent with previous studies45, and reaction products were analyzed by western blotting with anti-TAP antibodies. As a loading control, we used antibodies that recognize the Rpl3 protein from the large ribosomal 60S subunit. Strikingly, we found that BBV-seq, but not the linker or reverse BBV sequences, drove expression of TAP-RLuc to levels comparable with IRES-controlled protein synthesis  at position − 1 relative to the start codon AUG with a purine (A)  pyrimidine and unstructured linkers  or T−38TTTC−34 with G−38GAAG−34  drop in Renilla luciferase activity when M1–4 mutants were used instead of wild-type BBV-seq, indicating a lower capacity to promote protein synthesis  indicated a lack of stable secondary RNA structures , the 40S subunit might skip this AUG and initiate translation from an AUG located downstream in the coding sequence. In that case, a shorter protein(s) would be generated. To address this possibility, we extracted proteins and RNA from the same samples that were analyzed with the luciferase assays. As expected, western blotting showed a strong TAP-RLuc protein signal in samples derived from the reaction programmed with RNA containing full-length BBV-seq  within BBV-seq that are essential for translation initiation, we next examined BBV-seq mutants containing deletions of different lengths Fig. a using cion Fig. b. Hypothseq Fig. c, lane 2seq Fig. b,c. To cnal Fig. d. The TATaken together, these data suggest that all 39 nucleotides of BBV-seq are required for efficient translation initiation of the protein reporter in the translationally-competent yeast cell-free system  and ORF2 (RLuc). To prevent post-termination ribosomes from reinitiating, we inserted a stable hairpin between the two ORFs upstream of BBV-seq. To rule out the possibility that putative internal entry on this short BBV sequence could be compromised sterically, we placed a spacer between the hairpin and the BBV sequence ppp(5′)G] cap analog (m7G) or kept them unmodified. Equal amounts of these RNAs were added to cell-free translation reactions, and the amounts of translation products were analyzed by quantitative western blotting. For this analysis, we used anti-TAP antibodies to detect full-length TAP-RLuc, with antibodies against ribosomal protein Rpl3 providing normalization of the values obtained in each lane. This double-antibody approach coupled with quantitative near-infrared secondary antibody detection allowed measurements of TAP-RLuc and Rpl3 amounts in the same reactions, thus minimizing experimental variability.Capping the 5ʹ-ends of newly synthesized transcripts with m7GpppN (m7G) protects mRNAs from rapid degradation in cells and enables recruitment of eIF4E in canonical cap-dependent translation initiationAs expected, no protein reporter translation was detected in reactions with uncapped RNA lacking BBV-seq Fig. a, lane 1Taken together, these data indicate that BBV-seq can improve translation efficiency in the context of cap-dependent initiation, suggesting that BBV-seq and the 5′-cap have additive stimulatory effects on translation initiation.AUGG-3ʹ) improves correct start codon selection. The mammalian Kozak sequence contains a purine in − 3 and G in + 4 positions (the A of the AUG codon is designated + 1)51. In S. cerevisiae, recent studies identified a strong requirement for an A present in the − 3 position relative to the start AUG codon54. To address the possibility of BBV-seq providing the optimum start codon context in cap-dependent translation, we generated a BBV-seq mutant that carries a − 3A to − 3C nucleotide substitution relative to the start codon  would influence BBV-seq-mediated translation. In fact, one defining feature of a CITE is functionality on uncapped or A-capped mRNAs31. As a control, we used RNA modified with a conventional m7G-cap. Consistent with our previous results  was placed under the control of the strong constitutive TEF promoter and integrated into the chromosomal HO locus. The incorporation of an extra copy of TIF4631 was verified by sequencing the resulting hybrid HO locus, and expression of the protein was further confirmed by western blotting using antibodies against the 6× His-tag fused to the N-terminus of the HO-integrated eIF4G1  and 883–952 (RNA-binding domain 3) in TIF4631 integrated into the HO locus  by the additional deletion of the RNA-binding domain 2 in the ain Fig. . Northerain Fig. .Together, these data support a mechanistic model in which BBV-seq-mediated translation initiation on uncapped mRNA involves recruitment of eIF4G1 to BBV-seq, likely via the RNA-binding domains of this factor.cis-acting RNA elements and trans-acting factors, ultimately resulting in the formation of elongation-competent 80S ribosomes positioned on the initiation codon of an mRNA4. During conventional translation initiation in eukaryotes, the eIF4F complex is recruited to the 5ʹ-UTR of mRNA prior to other steps of translation initiation. The eIF4E subunit of the eIF4F complex binds the m7G cap-modified 5ʹ-end of a transcript, while eIF4G interaction promotes recruitment of the RNA-helicase eIF4A to the mRNA. eIF4A unravels the secondary structure of the leader sequence, facilitating mRNA scanning by 43S PIC in search of the start codon. In this mechanism, eIF4G acts as a scaffolding protein  reporter mRNAs when BBV-seq was placed upstream of the reporter ORF  from the bicistronic reporter construct TAP-BBV-seq-RLuc  IRES; and to β-globin mRNA63. Thus, it seemed possible that eIF4G1 may directly interact with RNA leader sequences, the translation of which occurs via cap-independent mechanisms, constituting a prerequisite step for recruitment of other factors needed for translation initiation. This model predicts that Arg-rich RNA-binding domains that can bind RNA directly58 may promote eIF4G1 interaction with BBV-seq. RNA-binding domain(s)-deleted strains are translationally impaired, which is somewhat difficult to address in cellular systems. However, the generated PTEF-6× HIS-TIF4631 strain allowed us to overcome this experimental obstacle. First, PTEF-6× HIS-TIF4631 demonstrates increased translational efficiency over the wild-type strain  eIF4G1 and eIF4A interact directly64, (ii) eIF4G1’s RNA-binding domains 2 and 3 do not overlap with its eIF4A-binding domain  besides unraveling RNA structure, eIF4A was implicated in providing directionality to the 43S complex during scanning, thereby decreasing the average dwell time of 43S on the 5′-UTR49. Another possibility might be that yeast eIF4G1 binds leader RNA via its HEAT domain similarly to mammalian eIF4G66, while the arginine-rich sequences previously annotated as RNA-binding domains 2 and 3 (examined here) may confer correct protein conformation. In this scenario, eIF4G1Δ2Δ3 might bind BBV-seq via the HEAT domain but in an unproductive fashion, competing with endogenous wild-type eIF4G1 for interactions with 5′ UTR. In fact, mammalian eIF4G’s HEAT domain binds specifically to EMCV and other picornavirus IRESs65 as well as to eIF4A, and an adjacent domain binds to β-globin mRNA and has been implicated in scanning63. Future experiments are needed to distinguish between these two models by exploring details of eIF4G1 binding to BBV-seq RNA. Regardless, the results presented here support a model in which eIF4G1 captures BBV-seq RNA, facilitating 43S PIC recruitment and translation initiation 30. It was found that EMCV-J-K leader sequence acts as a CITE element that directly binds eIF4G and promotes translation initiation by a mechanism that requires a free 5′-end and involves ribosomal scanning. Thus, the CITE mechanism is distinct from IRES-mediated translation initiation, as it depends on 5ʹ-end availability and functions upon modification of the 5′-end with an A-cap. In many cases CITEs do not require a full set of translational factors31. Finally, a recent study conducted in mammalian cells has revealed the requirement for a single-stranded and unstructured A-rich sequence for cap-independent translation initiation69. All these characteristics of CITE are inherent to BBV-seq dissected in this work  and DNA concentrations were estimated spectrophotometrically. To synthetize uncapped RNA, 1 μg of PCR-generated DNA template was transcribed in the T7-polymerase reaction using TranscriptAid T7 High Yield Transcription kit (Thermo Scientific) according to the manufacturer's recommendations. Reaction mixtures were incubated for 120 min at 37 °C, followed by DNase treatment for 15 min at 37 °C. RNA transcripts were first purified by phenol/chloroform extraction, followed by EtOH precipitation; pellets were resuspended in 50 μL of H2O and separated from unincorporated nucleotides by gel filtration on Micro Bio-Spin P-30 columns (Bio-Rad). RNA was aliquoted and stored at − 80 °C.PCR reactions were performed with DreamTaq (Thermo Fisher) in 50 μL reaction volume using 10 ng of a pYes plasmid construct as a template, 0.4 μM forward (5′-CGGATCGGACTACTAGCAGCTG -3′) and 0.4 μM reverse (5′-TTCATTAATGCAGGGCCGCAAATT-3′) primers that anneal upstream and downstream of the To generate capped RNA, purified PCR products (described above) were used as DNA templates in coupled T7 RNA polymerization–RNA capping reactions , incubated for 1 h at 37 °C.https://rna.urmc.rochester.edu/RNAstructureWeb/).We predicted putative secondary structures and their respective ΔG values  for wild type or mutant RNA sequences using the "Predict a Secondary Structure” server (RNA structure tools version 6.0.1) (70. Gels were stained with SYBR Gold (Invitrogen) and scanned using a Typhoon 9200 imager  at 532 nm to visualize RNA. RNA was then transferred onto nylon membrane and hybridized with a [32P]-labeled probe specific for the gene encoding TAP (5′-GCCGAATTCTCCCTGAAAA-3′) or with [32P]-labeled probe y540 against 25S rRNA (5′-TCCTACCTGATTTGAGGTCAAAC). Radioactive signal was detected using Typhoon 9200 in the phosphorimaging mode and analyzed using ImageQuant software . For quantification, volume of the hybridization signal corresponding to the RNA species of interest was converted to phosphorimaging units, and the background (average image background) was subtracted. For normalization of TAP RNA, the phosphorimaging units corresponding to this RNA were divided by that derived from the 25S rRNA signal present in the same sample.RNA was separated on 1.2% formaldehyde-containing agarose gel as described previouslySaccharomyces cerevisiae cells grown in 1 L of YPD medium to OD600 ~ 0.5 were harvested, washed, incubated at room temperature in 1 M sorbitol, then in YPD. Cells were collected, washed in buffer A (20 mM Hepes–KOH (pH 7.4), 100 mM KOAc, 2 mM Mg(OAc)2, 2 mM DTT) and the pellet was resuspended in buffer A containing 25% mannitol and 0.1 mM PMSF in 2:3 volume/cell weight ratio. The cell slurry was dripped directly into liquid nitrogen to form small ice beads. Frozen yeast/buffer beads were powdered in a SPEX freezer mill chamber. The powdered yeast cells were thawed and centrifuged in a Beckman ultracentrifuge for 15 min at 30,000×g using a fixed angle Beckman rotor Type 80 Ti. The clear phase was collected (~ 4 mL), centrifuged for 30 min at 100,000×g using the same rotor and the supernatant was collected. 2.5 ml of supernatant was applied on gel filtration columns G-25  and 5 mL of buffer A containing 10% glycerol was used for elution. The RNA content in eluted fractions was determined spectrophotometrically; fractions with at least 75% of the highest RNA concentration were pooled, aliquoted and frozen in liquid nitrogen for storage at − 80 °C.43, with a few modifications. In brief, for one reaction we used 7.5 μL of yeast extract , 2 μL of RNA (100–300 ng), 3 μL of 5 × translation buffer (100 mM Hepes–KOH (pH 7.6), 50 μM each amino acid, 10 mM Mg(OAc)2, 250 mM KOAc, 100 mM creatine phosphate, 0.6 U/μL creatine phosphokinase, 10 mM DTT, 5 mM ATP, 0.5 mM GTP, 15 U RiboLock, 5× SIGMAFAST Protease Inhibitor Cocktail), 2.5 μL of H2O. The total reaction volume was 15 μL. Reactions were incubated at 21 °C for 90 min. Prior to reaction, RNA was folded. The folding reactions containing 10 μg of mRNA transcript in 12 μL of H2O were heated at 95 °C for 2 min, cooled on ice for 2 min, mixed with 6.6 μL folding buffer  and incubated at 37 °C for 30 min71. We found no differences in protein yield between folded or unfolded mRNA containing BBV-seq; therefore, we omitted the RNA folding step when BBV-seq-containing RNAs were included in cell-free translation reactions.The translation reactions were carried out as described previouslyTo analyze in vitro translation reaction products, proteins were isolated from 15 μL of the translation reactions using TRI REAGENT-LS according to the manufacturer’s recommendations. Protein pellets were resuspended in 50 μL of 1xSDS-PAGE loading dye, and 10 μL were analyzed by SDS-PAGE using 10% polyacrylamide gels in duplicate. To analyze expression of 6xHis-eIF4G1 and its RNA-binding domain deletion mutants, the corresponding yeast strains were lysed in Buffer A supplemented with PMSF and SIGMAFAST Protease Inhibitor Cocktail by glass-beads shearing. The lysates were clarified by centrifugation and a total of 10 μg protein, as measured by the Bradford assay, was separated by SDS-PAGE using 8% polyacrylamide gels. Proteins were transferred onto nitrocellulose membrane and Ponceau S stained to verify equal loading. For conventional western blotting with ECL detection, we used 10% milk in PBS—0.1% Tween 20 (PBST) for blocking (20 min), and PBST for washing. For quantitative western blotting, Ponceau S staining was omitted; prior to blocking, membranes were dried at 37 °C for 15 min, rehydrated and blocked with 5% milk in PBS for 60 min. For detection, membranes were dried at 37 °C for 30 min and kept in a dark chamber. We used IR-Dye-680RD secondary antibodies for Rpl3-probed blots, and IR-Dye-800CW antibodies for TAP-probed blots. IR signals were detected on a Typhoon imager by scanning membranes at 800 nm and 680 nm; ImageQuant was used to analyze the images. For normalization, bands corresponding to TAP-RLuc and Rpl3 were converted to phosphorimager units and TAP-RLuc/Rpl3 ratios were plotted as bar graphs.Renilla Luciferase Assay System from Promega (cat#E2810) according to the manufacturer's protocol. The luminescent signal was measured on a GLOMAX 20/20 luminometer. Statistical analysis was performed by one-way ANOVA with GraphPad PRISM 5.We used the For RNA and protein extraction from the luciferase reaction, 100 μL of TRI REAGENT-LS reagent were added to 100 μL of the luciferase reaction. Samples were stored at − 80 °C prior to processing according to the manufacturer’s recommendations.Supplementary Information.


DEAD-box helicases are a large family of conserved RNA-binding proteins that belong to the broader group of cellular DExD/H helicases. Members of the DEAD-box helicase family have roles throughout cellular RNA metabolism from biogenesis to decay. Moreover, there is emerging evidence that cellular RNA helicases, including DEAD-box helicases, play roles in the recognition of foreign nucleic acids and the modulation of viral infection. As intracellular parasites, viruses must evade detection by innate immune sensing mechanisms and degradation by cellular machinery while also manipulating host cell processes to facilitate replication. The ability of DEAD-box helicases to recognize RNA in a sequence-independent manner, as well as the breadth of cellular functions carried out by members of this family, lead them to influence innate recognition and viral infections in multiple ways. Indeed, DEAD-box helicases have been shown to contribute to intracellular immune sensing, act as antiviral effectors, and even to be coopted by viruses to promote their replication. However, our understanding of the mechanisms underlying these interactions, as well as the cellular roles of DEAD-box helicases themselves, is limited in many cases. We will discuss the diverse roles that members of the DEAD-box helicase family play during viral infections. The broad family of DExD/H helicases, including DEAD-box helicases and the related DEAH-box helicases, belongs to superfamily 2 of the RNA helicase family. This family is characterized by a series of amino acid motifs that form the RNA and ATP binding sites of the helicase core ,2; DEAD-In vitro studies of DEAD-box helicase unwinding activity have revealed ATP-dependent RNA helicase function, but these proteins tend to have only weakly processive helicase activity, and instead are thought to promote local RNA:RNA rearrangements . Beyond Although DEAD-box helicases share a conserved core domain, variable N- and C-terminal regions allow members of this protein family to act on diverse targets. These flanking domains target the helicases to particular RNAs or proteins; the diversity of these regions accounts for the breadth of function observed within this protein family . As a reInnate immune defenses depend upon the recognition of invading pathogens by the host, and viral infections, including RNA viruses, are largely sensed through nucleic acid recognition. RNA viruses present a host cell with various foreign RNA features, such as unique ends, structured RNA elements, or dsRNA replication intermediates, which can be recognized as hallmarks of infection. RNA binding proteins thus play important roles in the sensing of viral infection, and DExD/H helicases are well-suited to recognize structural features characteristic of viral RNAs. The canonical cytoplasmic sensors of viral RNA, the RIG-I-like receptors (RLRs) RIG-I and MDA-5, contain DExD/H-box helicase domains . RIG-I rDEAD-box helicases have been shown to contribute to cytosolic viral RNA recognition and to feed into these canonical immune signaling pathways, sometimes acting in concert with DEAH-box helicases. Several DEAD-box helicases promote interferon induction or other inflammatory signaling. DDX1 contributes to IFN-β activation during transmissible gastroenteritis virus infection via its interaction with the nonstructural protein nsP14  and inteCytosolic DNA recognition represents another important surveillance mechanism for stimulating innate immune pathways in response to invasion by DNA viruses and bacteria, as the cytoplasm has a dearth of DNA under normal conditions. Much of this signaling is now known to occur via the cGAS-STING pathway, where DNA recognition by cGAS leads to the production of the cyclic dinucleotide cGAMP, which signals through STING, leading to phosphorylation of IRF3 by TBK1 ,24. HoweListeria monocytogenes infection . St. St39]. nd DDX56 ,41.Arabidopsis homolog of DDX17, RH30, has also been shown to be antiviral against tombus viruses such as tomato bushy stunt virus and cucumber necrosis virus, both in plant hosts and a yeast surrogate host model [DDX17 and DDX5 are nuclear resident paralogs that have diverse functions in transcription, splicing, miRNA biogenesis, mRNA export, and ribosome biogenesis . DDX17 bst model . RH30 rest model . In contst model . DDX5 isst model . DDX3 isst model .DDX23 is a nuclear resident helicase with known roles in splicing. Studies in the invertebrate chordate amphioxus identified DDX23 as a dsRNA binding protein, and found that dsRNA binding was conserved in human cells . How DDXThe nucleolus is the site of ribosomal RNA (rRNA) transcription, rRNA biogenesis, and assembly of ribosomal subunits. Nucleoli form as sites of concentrated processing factors around clusters of rDNA genes, and liquid–liquid phase separation drives the organization of subcompartments in which rRNA is cleaved and modified ,50. ManyThere is a growing appreciation for the role of the nucleolus in cellular stress responses, including responses to viral infections ,52. KnowDDX21 is another nucleolar helicase with cellular functions in ribosomal RNA production  that alsTaken together, these examples paint a picture where nucleolar proteins are influenced by, and exert effects on, cytoplasmic processes.P bodies are nonmembranous organelles in the cytosol that contribute to the regulation of cellular RNAs—resident RNAs can be stored, translationally repressed, and/or degraded . Many viDDX6 has been shown to have pro- or anti-viral roles in a variety of viral infections, including several arthropod-borne viruses. DDX6 enhances RIG-I signaling, interacting with both RIG-I and influenza viral RNA in infected cells . HoweverViruses can also hijack P-body components to aid in replication. DDX6 and other P body components have been found by mass spectrometry to bind the dengue virus 3′ UTR RNA, but in constrast to other flaviviruses, DENV infection is attenuated by DDX6 knockdown . West NiStress granules are another type of nonmembranous organelle which form when translation is impaired . The strDDX1 is another stress granule resident protein  and has These interactions highlight the ways that both viruses and hosts can use RNP interactions to promote or antagonize one another’s function . The abiThe anti-viral and pro-viral DEAD-box helicase relationships that have been described so far in the literature likely represent only the tip of the iceberg in terms of the multitude of roles that DEAD-box helicases play in innate immunity and the modulation of viral infection . These rAlthough much remains to be defined about the antiviral roles of the DEAD-box helicase family, some broad themes have emerged. First, the functions of DEAD-box helicases in viral infection appear to often, but not always, rely on direct interactions between the host DEAD-box protein and viral RNAs. Some DEAD/DEAH-box helicases, such as the RLRs RIG-I and MDA-5, are specialized innate immune sensors, and others, such as DDX3X, have cellular roles but also contribute to innate immune signaling pathways. Still others bind viral RNA as antiviral effectors, whether induced by interferon signaling in the case of DDX60L or independently of known innate immune pathways, as is the case for DDX17 and DDX56. Continuing to biochemically define the RNA features recognized by antiviral helicases and the ways those RNA structures may be altered by helicase activity could help us identify functionally-important RNA structures in viral replication. Additionally, in recent years, the application of RNA probing technologies such as SHAPE to viral RNAs is beginning to enable detailed analysis of secondary and tertiary structures that may be functionally relevant ,91,92. ASecond, DEAD-box helicase/virus interactions are highly context-dependent: different viruses are affected in different ways by the same DEAD-box helicases, and although RNA binding is important in many cases, there also exist examples where helicase function is dispensable for the antiviral activities that have been characterized. Detailed mechanistic studies naturally lag far behind the high-throughput genomic and proteomic identification of virus-host interactions, but a deeper investigation of the virus and host determinants underlying antiviral DEAD-box helicase function may illuminate key characteristics governing the outcome of a virus-helicase interaction.Third, our understanding of the antiviral function of DEAD-box helicases is limited by a lack of information about their cellular roles. DEAD-box helicases are essential for many aspects of RNA biogenesis, but in many cases the details of their roles in these pathways have yet to be fully elucidated, and specific targets, activities, and cofactors are not well defined. Our limited knowledge of DEAD-box helicase function in both contexts hampers our ability to understand determinants governing RNA substrate selection and helicase activity and certainly our ability to predict whether or how they might interact with viral infection. We speculate that, in many cases, the viral substrates of these proteins might resemble their endogenous targets, whether through commonalities in target RNA secondary structure or in interactions with the variable N- and C-terminal domains that disrupt or coopt their cellular roles. As further examples and details of DEAD-box helicases in viral infection are characterized, complementary studies of their normal cellular functions will be necessary to determine how their interactions with viral infection resemble or differ from their normal biology. Ultimately, a better understanding of the virus–host interactions that affect the outcome of infection could lead to better antiviral therapeutics. Virus research has a long history of providing insight into cellular function, and these mechanisms will also teach us about cellular RNA regulation in broader contexts. This is an exciting time to explore the interface between viruses and cellular helicases.


Finally, we compared our optimized RNA extraction and RT-qPCR protocol with current best-practice immune assays and demonstrated that our assay is a sensitive alternative to protein-based assays for peptide-specific responses, especially with limited PBMCs number. This protocol with high analytical and diagnostic sensitivity has broad applicability for both primary research and clinical practice.Reliable extraction and sensitive detection of RNA from human peripheral blood mononuclear cells (PBMCs) is critical for a broad spectrum of immunology research and clinical diagnostics. RNA analysis platforms are dependent upon high-quality and high-quantity RNA; however, sensitive detection of specific responses associated with high-quality RNA extractions from human samples with limited PBMCs can be challenging. Furthermore, the comparative sensitivity between RNA quantification and best-practice protein quantification is poorly defined. Therefore, we provide herein a critical evaluation of the wide variety of current generation of RNA-based kits for PBMCs, representative of several strategies designed to maximize sensitivity. We assess these kits with a reverse transcription quantitative PCR (RT-qPCR) assay optimized for both analytically and diagnostically sensitive cell-based RNA-based applications. Specifically, three RNA extraction kits, one post-extraction RNA purification/concentration kit, four SYBR master-mix kits, and four reverse transcription kits were tested. RNA extraction and RT-qPCR reaction efficiency were evaluated with commonly used reference and cytokine genes. Significant variation in RNA expression of reference genes was apparent, and absolute quantification based on cell number was established as an effective RT-qPCR normalization strategy. We defined an optimized RNA extraction and RT-qPCR protocol with an analytical sensitivity capable of single cell RNA detection. The diagnostic sensitivity of this assay was sufficient to show a CD8 Reliable isolation of high quality and high quantity RNA from peripheral blood mononuclear cells (PBMCs) and other cells is critical for a broad range of basic, preclinical, and clinical applications , 2. RNA-A range of RNA-based platforms are now available, all dependent upon high quality and high quantity RNA . Howeverin-situ hybridization, RNA microarrays etc.) (Reverse transcription quantitative PCR (RT-qPCR) remains the “Gold Standard” for assay of gene expression as an alternate readout to protein expression , 37. RT-ys etc.) –40. Moreys etc.) , 42 but ys etc.) , 44. In in vitro stimulation has been shown to modulate the expression of many commonly used reference genes , (Sigma-Aldrich)- Ionomycin (Iono), (Sigma-Aldrich)- Phytohemagglutinin-L (PHA), (Sigma-Aldrich)- Human Cytomegalovirus, Epstein Barr Virus and Influenza virus Synthetic peptides .- ssoAdvanced™ Universal SYBR® Green Master-Mix (Bio-Rad)- QuantiNova SYBR® Green PCR Kit (QIAGEN).- PowerUp SYBR® Green Master-Mix (Applied Biosystems)2 SYBR® Green qPCR Master-Mix (QIAGEN).- RT- RNeasy® Mini Kit (QIAGEN)- RNeasy® MiniElute Cleanup Kit (QIAGEN)- RNeasy® Micro Kit (QIAGEN)mirVana™ Total RNA Isolation Kit (Applied Biosystems).- MagMAX™ - SuperScript™ III First-Strand Synthesis System (ThermoFisher)- SuperScript™ IV First-Strand Synthesis System (ThermoFisher)- iScript™ Advanced cDNA Synthesis Kit (Bio-Rad)- High-Capacity RNA-cDNA Kit™ (Applied Biosystems).- PrimerBank™ primers .- anti-human IFN-γ monoclonal antibody (mAb) - anti-human IFN-γ biotinylated mAb - anti-human IFN-γ-FITC mAb .- QuantStudio 3 Real-Time PCR system (Applied Biosystems)- NanoDrop 2000 Spectrophotometer (ThermoFisher)- 2100 Bioanalyzer (Agilent Technologies)- AID ELIspot reader system - LSRFortessa X-20 (BD Biosciences).- QuantStudio Design and Analysis Software - ProcartaPlex Analyst Software - FlowJo Software - GraphPad Prism .n = 12) provided by the Australian Red Cross Blood Service, under a protocol approved by the James Cook University Human Research Ethics Committee (#H6702). PBMCs were isolated by density gradient centrifugation and cryopreserved in FBS 10% DMSO. Prior to use, samples were thawed rapidly at 37°C, treated with DNAase I , and rested for 18 h at 2 × 106 cells/mL in media  at 37°C and 5% CO2. Viable PBMCs were counted prior to downstream analysis.Blood was collected from healthy donors or buffy coats (Genomic DNA was isolated from PBMCs using the QIAamp DNA Mini Kit (QIAGEN) according to manufacturer's instructions. High-resolution class I and class II HLA typing was performed by the Australian Red Cross Transplant and Immunological Services  using the MIA FORA NGS FLEX HLA typing kit (Immunocor) and Illumina MiSeq and MiniSeq platforms.−5 M β-Mercaptoethanol (Sigma-Aldrich) (complete media). Synthetic peptides (10 μg/mL) representing defined CD8+ T cell epitopes from human Cytomegalovirus, Epstein Barr Virus or Influenza virus  or at 1 × 106 cells/mL in 3 mL in 12-well flat-bottom plates (flow cytometry).PBMCs were resuspended in RPMI-1640 supplemented with 10% human serum, 100 U/mL penicillin/streptomycin, 2 mM glutaMAX (ThermoFisher Scientific), 10 mM HEPES (ThermoFisher Scientific), and 5 × 10za virus  were tesqPCR was conducted using the QuantStudio 3 Real-Time PCR system running QuantStudio Design and Analysis Software . A standard curve, combined calibration sample, and no template negative controls were included on each plate. All samples were run in technical triplicate in accordance with MIQE guidelines . cDNA sy2 SYBR® Green qPCR Master-Mix (QIAGEN). RPL13a, SDHA, TBP, and IFN-γ primer , QuantiNova SYBR® Green PCR Kit (QIAGEN), PowerUp SYBR® Green Master-Mix (Applied Biosystems) and RTγ primer  ampliconr 750 nM .6 unstimulated PBMCs using the RNeasy® Mini Kit (QIAGEN). Seven microliters of extracted RNA was converted to cDNA using the SuperScript™ III First-Strand Synthesis System kit (Invitrogen). Master-mix reaction efficiency was calculated from log10 diluted cDNA (104-101 cells/reaction) with RPL13a, SDHA, TBP, and IFN-γ primers at 500 nM.The four SYBR master-mix kits were further evaluated with efficiency titrations of cDNA standards. Briefly, RNA was extracted from 1 × 106 PBMCs were stimulated for 6, 12, 16, 24, or 48 h with or without PMA/Iono as described above. RNA was extracted with RNeasy® Mini Kit (QIAGEN). Seven microliters of extracted RNA was reverse transcribed with SuperScript™ III First-Strand Synthesis System kit (Invitrogen). qPCR was run with ssoAdvanced™ master-mix, RPL13a, SDHA, TBP and IFN-γ primers at 500 nM and samples at 102 cells/reaction.1 × 10mirVana™ Total RNA Isolation Kit (Applied Biosystems); and one post-extraction RNA purification and concentration kit, RNeasy® MiniElute Cleanup Kit (QIAGEN), were evaluated. All extractions included genomic DNA removal. RNA was extracted from 1 × 106 PBMCs incubated for 6 h with or without PMA/Iono, with the exception of the RNeasy® Micro Kit where 0.5 × 106 PBMCs was used (per manufacturer's recommendation). To evaluate concentration, kit eluates were concentrated using the RNeasy® MiniElute. All elutions were performed in the smallest recommended volume. The yield of extracted RNA was quantified using a NanoDrop 2000 Spectrophotometer (ThermoFisher). RNA yield , calculated RNA quality was assessed by RNA integrity number (RIN) by the Australian Genome Research Facility  using a 2100 Bioanalyzer (Agilent Technologies). Subsequently, 7 μL of RNA was converted to cDNA using the SuperScript™ III First-Strand Synthesis System kit (Invitrogen) alongside negative reverse transcriptase controls. qPCR was run with ssoAdvanced™ master-mix, RPL13a, SDHA, TBP, and IFN-γ primers at 500nM and sampled at 102 cells/reaction.To evaluate RNA yield and quality, three RNA extraction kits: RNeasy® Mini Kit (QIAGEN), RNeasy® Micro Kit (QIAGEN), and MagMAX™ mirVana™ Total RNA Isolation Kit (MagMAX) with or without the RNeasy® MiniElute Cleanup Kit. Briefly, 1 × 106 PBMCs were incubated for 6 h with or without PMA/Iono as described above. Seven microliter of RNA extracted by MagMAX was used for each cDNA synthesis kit. Alternatively, the maximum recommended input of RNA extracted by MagMAX in association with the RNeasy® MiniElute Cleanup Kit was used . qPCR was run with ssoAdvanced™ master-mix, RPL13a, SDHA, TBP, and IFN-γ primers at 500 nM and sample diluted to 102 cells/reaction; except when considering concentration, when the samples were run undiluted.Four reverse transcription kits were evaluated: SuperScript™ III First-Strand Synthesis System (ThermoFisher Scientific), SuperScript™ IV First-Strand Synthesis System (ThermoFisher), iScript™ Advanced cDNA Synthesis Kit (Bio-Rad) or High-Capacity RNA-cDNA Kit™ (Applied Biosystems). Kits were evaluated using RNA extracted using the MagMAX™ 10 serial dilution of unstimulated PBMCs (106-100 cells/extraction), using the MagMAX kit with or without the RNeasy® MiniElute Cleanup Kit. A media-only extraction control was processed in parallel. For determination of diagnostic sensitivity, RNA was extracted using the MagMAX kit from titrated PBMCs  incubated for 6 h with or without PMA/Iono or HLA-matched peptide. For sensitivity evaluations, 10 μL of RNA was converted to cDNA using the SuperScript™ IV First-Strand Synthesis System (Invitrogen). qPCR used undiluted sample with ssoAdvanced™ master-mix, RPL13a, SDHA, TBP, and IFN-γ primers at 500 nM.For determination of analytical sensitivity, RNA was extracted from a log5 PBMCs were plated in triplicate onto 96-well multi-screen filtration plates  pre-coated with anti-human IFN-γ monoclonal antibody (mAb)  and stimulated for 24 h with or without peptide, PMA/Iono, PHA or media. After washing, IFN-γ secreting cells were stained with 1 μg/mL of anti-human IFN-γ biotinylated mAb  followed by streptavidin alkaline phosphatase (MABTECH). The assay was developed using the AP Conjugate Substrate Kit (BioRad). IFN-γ-spot-forming cells were counted using AID ELIspot reader system .IFN-γ ELIspot assays were performed as previously described , 52. Bri5 PBMCs incubated for 6 h with or without peptide, PMA/Iono or media. Fifty microliter supernatant was analyzed using the ProcartaPlex Immunoassay (ThermoFisher) per manufacturer's protocol. Cytokine concentration was calculated from a standard curve using the ProcartaPlex Analyst 1.0 Software (ThermoFisher).Supernatant was collected from 4 × 106 PBMCs were incubated for 6 h with or without peptide, PMA/Iono or media. 5 μg/mL brefeldin A (BD Biosciences) was added after 1 h. Cells were then stained with Fixable Viability Stain 510 (BD Bioscience) and permeabilizated with the Cytofix/Cytoperm kit (BD Biosciences) before staining with anti-human IFN-γ-FITC  mAb. Data were acquired on a LSRFortessa X-20 driven by FACSDiva software (BD Biosciences) and analyzed using FlowJo software (version 10.4).3 × 10P < 0.05 were considered significant.RT-qPCR, Bioanalyzer and NanoDrop data were analyzed using a repeated-measures two-way ANOVA with Bonferroni-corrected multiple comparisons test comparing test to control mean. Correlation between RT-qPCR and protein quantification was test with linear regression analysis. Analysis was conducted using GraphPad Prism version 7.0 (GraphPad). In all cases, 2 SYBR® Green qPCR Master-Mix (QIAGEN)—were evaluated using two methods of preparing reference standards: (i) standards derived from log10 diluted amplicon; and (ii) standards generated from log10 diluted cDNA (RPL13a), moderate  and low  expression; and one set targeted a cytokine gene  , the largest dynamic range (100−4 copies/reaction), and the lowest mean deviation from 100% (R2 ≥ 0.97) were consistent for all primers tested, at all concentrations. Together, these data identify ssoAdvanced™ master-mix as providing the highest reaction efficiency for qPCR from PBMC cDNA.Four master-mixes—ssoAdvanced™ Universal SYBR® Green Master-Mix (Bio-Rad), QuantiNova SYBR® Green PCR Kit (QIAGEN), PowerUp SYBR® Green Master-Mix (Applied Biosystems) and RTted cDNA . Reactio; IFN-γ) . When co; IFN-γ)  and 43.8; IFN-γ)  failed. rom 100% . The coeRPL13a, SDHA, and TBP, previously reported as stable in PBMCs following stimulation  Total RNA Isolation (Applied Biosystems) kits were tested for 1) RNA yield and 2) concentration with or without a post-extraction RNA concentration step using the RNeasy® MiniElute Cleanup Kit (QIAGEN). In each case, PBMCs were incubated with or without PMA/Iono for 6 h. RIN assessment demonstrated that RNA integrity was high (>7) and consistent across all kits (6 cells): 0.87 vs. 1.36, P < 0.05 and 0.82 vs. 1.42, P < 0.01, respectively)   and MagMAX™ RPL13a, SDHA, TBP as well as IFN-γ quantified by RT-qPCR. Data were normalized to either RNA yield (copies/106 cells) or concentration (copies/μL). When considering both yield and concentration, RT-qPCR signal was significantly affected by extraction technique  and PMA/Iono , stimula6 cells: 1.1 × 107 vs. 1.3 × 107P < 0.05 SDHA; 4.1 × 106 vs. 5.6 × 106, P < 0.001 TBP; 6 cells: 5.8 × 108 vs. 6.9 × 108, P < 0.001 IFN-γ; 5.9 × 108 vs. 6.8 × 108, P < 0.05 RPL13a; 3.5 × 106 vs. 4.5 × 106, P < 0.05 TBP;7 vs. 2.5 × 107, P < 0.05 RPL13a; 7 vs. 3.4 × 107, P < 0.05 IFN-γ; 1.1 × 107 vs. 2.2 × 107, P < 0.001 RPL13a;Figure 3B) PBMCs. When assessing technical reproducibility, extraction had no statistically significant effect on variation between replicate extractions , iScript™ Advanced (Bio-Rad) and High-Capacity (Applied Biosystems) kits were tested in conjunction with MagMAX  and MagMractions . These d10 serial dilution of unstimulated PBMCs and expression of IFN-γ, RPL13a, SDHA, and TBP determined by absolute quantification. The highly expressed RPL13a gene was detected at single-cell level from both MagMAX  was driven by increased RT-qPCR signal at 105 and 104 PBMCs per extraction. These data establish that our optimized protocol is capable of extracting and quantifying RNA of a highly expressed gene from a single cell. Importantly, the additional step of RNeasy® MiniElute Cleanup did not further enhance analytical sensitivity.We next evaluated the analytical sensitivity of our optimized protocol using MagMAX extraction kit  and MagM+ T cell epitopes restricted by different MHC molecules, quantifying IFN-γ production by flow cytometry, cytokine bead array and ELIspot; and IFN-γ mRNA by our optimized protocol. A limited epitope-specific IFN-γ response was demonstrated by flow cytometry ; P = 0.0002, R2 = 0.7593 (1 × 105); P = 0.0028, R2 = 0.6065 (2.5 × 104); P = 0.0058, R2 = 0.5493 (1 × 104); P = 0.0056, R2 = 0.5520 (4 × 105); P = 0.0054, R2 = 0.5548 (1 × 105); P = 0.0138, R2 = 0.4707 (2.5 × 104); P = 0.0050, R2 = 0.5617 (1 × 104); 4 cells, representing a clinically and diagnostically meaningful reduction in cell number.Next, we assessed correlation between results obtained using our optimized RT-qPCR protocol and current best-practice immunoassays. The correlation between our RT-qPCR protocol and ELIspot was significant at all cell numbers tested [4 cells, and represents a sensitive and robust alternative to protein quantification for research, diagnostic and clinical applications.Taken together, we report here a highly sensitive RNA extraction and RT-qPCR quantification strategy using the MagMAX RNA extraction kit, Superscript™ IV reverse-transcription kit and ssoAdvanced™ SYBR master-mix . This asHerein, we describe an optimized RNA extraction and RT-qPCR protocol requiring low PBMC numbers, with high analytical and diagnostic sensitivity, whilst maintaining high correlation to protein-level quantification that is typically reliant on much larger cell numbers for detection.Precise RT-qPCR results are typically dependent on reactions maintaining efficiency close to 100% . Both asWe tested three RNA extraction kits by evaluating extraction quality and efficiency: RNeasy® Mini Kit, RNeasy® Micro Kit, and MagMAX™ mirVana™ Kit; in combination with the RNA purification and concentration kit: RNeasy® MiniElute Cleanup Kit. When extracting identically controlled samples, all kits yielded RNA with equivalent RIN scores and low technical variability between replicates. Importantly, RNA yield from PBMCs was significantly increased using MagMAX as compared to silica-column technologies. Additionally, when compared to silica-column extractions, we found MagMAX was more cost and time efficient when running larger number of samples . We therefore expect magnetic bead-based extractions will become increasingly common within blood-based nucleic acid isolations , 61, 62.Four reverse transcriptase (RT) kits were also evaluated: SuperScript™ III First-Strand Synthesis System, SuperScript™ IV First-Strand Synthesis System, iScript™ Advanced cDNA Synthesis Kit and High-Capacity RNA-cDNA Kit™. Of those, Superscript™ IV was associated with the highest qPCR signal. A previous study evaluating RNA extracted from PBMCs using earlier-generation RT kits reported >128-fold increased qPCR signal between kits . This work will significantly improve analytical capacity of studies relying on irreplaceable, relatively small or costly human samples   , 67.RPL13a, SDHA and TBP following stimulation. This is not unexpected as reference genes have been described as variable across cell types, tissues, and experimental and stimulatory conditions (Another important outcome of our work is the finding that absolute quantification of transcripts and subsequent normalization to cell numbers is the most appropriate analysis strategy for RNA/RT-qPCR quantification from PBMCs , 48. We nditions , 68, 69.mirVana™ Total RNA Isolation Kit and reverse transcribed with SuperScript™ IV First-Strand Synthesis System. Our assay provided single cell analytical sensitivity and a diagnostic sensitivity that could define response hierarchy from 1 × 104 cells. This assay offers an alternative to current best practice protein-based immunoassays, especially for limited PBMC numbers. This work has broad applicability for both clinical and primary research practice.In summary, we report herein the development of an optimized PBMC RNA extraction and RT-qPCR protocol. We employed a qPCR strategy of absolute quantification utilizing PrimerBank™ primers and ssoAdvanced™ Universal SYBR® Green Master-Mix. PBMC RNA was isolated with MagMAX™ The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The studies involving human participants were reviewed and approved by James Cook University Human Research Ethics Committee. The patients/participants provided their written informed consent to participate in this study.DB and CL performed experiments. DB, JB, CL, and DD designed experiments. DB, JB, AW, CL, and DD analyzed and interpreted the data. DB, CL, and DD wrote the manuscript, with input from JB and AW.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.