Journal: eLife

Article Title: Origin of wiring specificity in an olfactory map revealed by neuron type–specific, time-lapse imaging of dendrite targeting

doi: 10.7554/eLife.85521

Figure Lengend Snippet:

Article Snippet: Using NotI-containing forward and KpnI-containing reverse primers, FRT-stop-FRT-myr-4xSNAPf was PCR amplified and subcloned into p10XQUAST. p10XQUAST was generated using p5XQUAS (Addgene #24349) and p10xQUAS-CsChrimson (Addgene #163629). attP24 and 86Fb landing sites were used for site-directed integration.

Techniques: Recombinant, Software

Journal: bioRxiv

Article Title: 3’ to 5’ Translation of Circular RNAs?

doi: 10.64898/2025.12.08.692888

Figure Lengend Snippet: (A) Schematic of vector design for backward translation of enhanced green fluorescent protein (eGFP) in human cells. The pcDNA3.1 vector was used as the backbone for circRNA expression. The construct consisted of two reverse eGFP fragments (coordinates: 593→1 and 720→594), separated by a translation initiation site (TIS)-containing sequence. The TIS sequence was flanked by a backward start codon (←GUA) and a backward stop codon (AAU←). TIS elements used in this study included either upstream sequences derived from endogenous backward ORFs or the EMCV internal ribosome entry site (IRES). (B) Structural illustration of two circ-(bw)-eGFP constructs: one lacking a TIS sequence element and one incorporating EMCV-IRES as TIS. The eGFP sequence is shown in green, and the TIS region is marked in red. (C) RT-qPCR quantification of reverse eGFP expression in HEK-293T cells transfected with empty vector, reverse eGFP without TIS, or reverse eGFP containing EMCV-IRES as TIS ( n =3). Tubulin was used as the internal reference gene. Primers spanning the back-splice junction (BSJ) were used to specifically detect circular RNA. All RNA samples were treated with RNase R to enrich for circular species. (D) Representative confocal microscopy images showing eGFP fluorescence (green) resulting from backward translation of circ-(bw)-eGFP in HEK-293T cells. Fluorescence was observed in cells transfected with reverse eGFP containing EMCV-IRES, but not in those lacking TIS. Scale bar, 20 μm. The fluorescent signal was detected in ∼1–2 out of every 100,000 cells. Live cells were stained with Mito-Tracker (red, mitochondria). (E) Summary of four sequence features known to promote circRNA translation upstream of backward ORFs: IRES-like elements predicted by IRESfinder, IRES-like hexamers aligned to the 10-nt region upstream of the backward start codon, Kozak consensus sequences identified by in-house scripts, and N6-methyladenosine (m⁶A) sites predicted using SRAMP. (F) The secondary structure of conserved motif ‘TCCCA…TGGGA’ identified from backward-translated circRNAs using MEME analysis. (G) Predicted secondary structures of circRNAs involved in regulating backward translation, modeled using RNAfold. From left to right: hs. circHSH2D; circ-(bw)-eGFP incorporating hs. circHSH2D-derived TIS; circ-(bw)-eGFP with the same TIS but with a stem-loop structure disrupted by mutation; and circ-(bw)-eGFP in which the backward start codon (5’-GUA-3’) was mutated to 5’-GCA-3’. The latter two constructs were predicted to be untranslatable. (H) RT-qPCR quantification of reverse eGFP in HEK-293T cells transfected with empty vector, hs. circHSH2D-TIS-(bw)-eGFP, hs. circHSH2D-TIS-(bw)-eGFP with stem-loop disruption, and hs. circHSH2D-TIS-(bw)-eGFP with start codon mutation (n=3). Tubulin was used as the internal control. Primers spanned the BSJ, and RNase R treatment was applied to enrich for circRNAs. (I) Representative confocal microscopy images showing eGFP fluorescence (green) from backward translation of circ-(bw)-eGFP in HEK-293T cells transfected with constructs listed in panel h. Fluorescence was observed only in cells transfected with hs. circHSH2D-TIS-(bw)-eGFP. No signal was detected in other groups. Scale bar, 20 μm. live cells were stained with Mito-Tracker (red, mitochondria). Statistical comparisons of eGFP expression levels under different conditions were performed using Mann-Whitney U in GraphPad Prism (version 9.2.0). Statistical significance: ns : not significant, “*”: p <0.05, “**”: p <0.01, “***”: p <0.001, “****”: p <0.0001.

Article Snippet: The plasmids for human cell lines in this study were generated using pcDNA3.1(+) ZKSCAN1 MCS + Sense IRES (Addgene: #69909) and pcDNA3.1(+).

Techniques: Plasmid Preparation, Expressing, Construct, Sequencing, Derivative Assay, Quantitative RT-PCR, Transfection, Confocal Microscopy, Fluorescence, Staining, Mutagenesis, Disruption, Control, MANN-WHITNEY

Journal: ACS synthetic biology

Article Title: Synthetic circuit-driven expression of heterologous enzymes for disease detection

doi: 10.1021/acssynbio.1c00133

Figure Lengend Snippet: A) To detect or monitor the presence of cancer cells, an RNA-based AND gate is used to express a heterologous biomarker (secreted TEV protease) that can be detected either in the urine or blood. In response to each of two cancer-associated transcription factors (TF1 and TF2), cognate SPECS (P1 and P2) drive the expression of input module 1 or 2, respectively (Input 1 and Input 2). Only when both input modules are triggered will the heterologous biomarker be expressed and secreted. B) To implement the synthetic circuit in vivo , three viruses, each bearing one of the input (1 or 2) or output gene modules, are intraperitoneally (i.p.) injected into tumor-bearing mice. Successful transduction leads to subsequent expression of the AND-gated heterologous reporter enzyme in cancer cells, in response to the cancer-associated transcription factors that provide the upstream circuit inputs. C) The sense-and-respond system can yield an activity-based signal to be read out either in the urine or in the blood. For the urine readout (top), mice were intravenously (i.v.) administered an in vivo nanosensor consisting of a reporter-tagged, TEV-specific peptide substrate conjugated to the surface of a poly(ethylene glycol) (PEG) carrier. When exposed to active TEV protease in vivo , reporter peptides are liberated from the TEV-sensitive nanosensor and accumulate into the urine where they are measured via immunoassay. For the blood readout (bottom), a quenched fluorescent reporter was designed to measure, ex vivo , the activity of TEV protease present in plasma. Cleavage by TEV proteases results in fluorescence dequenching and signal generation.

Article Snippet: For the output plasmid, synthetic promoter GAL4BS was placed upstream of transmembrane GFP (hTFR-GFP, Addgene plasmid 45060) or TEV protease (pcDNA3.1-V5-hTEV, Addgene plasmid 65800).

Techniques: Biomarker Discovery, Expressing, In Vivo, Injection, Transduction, Activity Assay, Ex Vivo, Clinical Proteomics, Fluorescence

Journal: ACS synthetic biology

Article Title: Synthetic circuit-driven expression of heterologous enzymes for disease detection

doi: 10.1021/acssynbio.1c00133

Figure Lengend Snippet: A) Input 1 relies on a SPEC (S( E2F1 )P) to drive the specific expression of endoribonuclease Cys4. Input 2 relies on a SPEC (S( cMyc )P) to drive the transcription of a fusion protein, GAD, consisting of the GAL4 DNA binding domain and the transcriptional transactivator VP16, and a miRNA transcript. The 3’ end of GAD transcripts has a hairpin structure for Csy4 binding and a miRNA binding site. When both inputs are present ([1,1] state), Csy4 will bind to the hairpin structure to release the inhibitory effect of the miRNA on GAD translation, driving the expression of GAD. The output expression is driven by a synthetic promoter GAL4BS, which is targeted by transcription activator-binding domain fusion, GAD. Two outputs are used in this study: membrane GFP (hTFR-GFP) or secreted TEV protease (SecTEV). B) OVCAR8 cells were either transduced with the output virus carrying a membrane GFP expression cassette ([0,0]), or the output virus with one of two input viruses ([1,0], [0,1]), or both input viruses and the output virus ([1,1]). After two days, cells were fixed for immunofluorescence imaging or flow cytometry. Scale bar: 100μm. C) Quantification of flow cytometry analysis of mean GFP fluorescent intensity with different input and output configurations. Mean ± s.d.; N=3; ordinary one-way ANOVA with Tukey’s correction for multiple comparisons, *** P =0.0007 for [1,1] vs. [0,0], ns P =0.5151 for [0,1] vs. [0,0], ns P =0.9717 for [1,0] vs. [0,0]. D) Human ovarian cancer cell lines (ES-2, OAW-42, CaOV-3, and OVCAR8), a hepatocarcinoma cell line (Huh7), and a cervical cancer cell line (Hela) were transduced with both input viruses and the output virus with GFP output ([1,1]). After two days, cells were fixed for flow cytometry analysis. The mean GFP fluorescent intensity is shown. Mean ± s.d.; N=3; unpaired two-tailed t-test, *** P =0.001 for ES-2, ** P =0.0015 for CaOV-3.

Article Snippet: For the output plasmid, synthetic promoter GAL4BS was placed upstream of transmembrane GFP (hTFR-GFP, Addgene plasmid 45060) or TEV protease (pcDNA3.1-V5-hTEV, Addgene plasmid 65800).

Techniques: Expressing, Binding Assay, Membrane, Transduction, Virus, Immunofluorescence, Imaging, Flow Cytometry, Two Tailed Test

Journal: ACS synthetic biology

Article Title: Synthetic circuit-driven expression of heterologous enzymes for disease detection

doi: 10.1021/acssynbio.1c00133

Figure Lengend Snippet: A) Purified protease enzymes or culture media from mammalian cells recombinantly expressing TEV protease were screened against a FRET-paired TEV substrate, and fluorescence activation was monitored over time. B) Kinetic fluorescence curves are shown for 1μM FRET-paired reporter incubated with TEV (20nM or 40nM) as well as MMP2, MMP9, MMP13, thrombin, and uPA (all 20 nM), or buffer without protease (Buffer). Mean ± s.d.; N=3. C, D) Activity of cellular wild-type TEV, secreted wild-type TEV (WT SecTEV), and secreted, unglycosylated TEV (SecTEV QSG), present in cell culture media, against FRET-paired TEV substrate, showing cleavage kinetics (C) and fluorescence fold changes at 30 minutes (D). Mean ± s.d.; N=2; unpaired two-tailed t-test, * P =0.0123 SecTEV QSG vs. Cellular TEV, * P =0.0106 for SecTEV QSG vs. WT SecTEV.

Article Snippet: For the output plasmid, synthetic promoter GAL4BS was placed upstream of transmembrane GFP (hTFR-GFP, Addgene plasmid 45060) or TEV protease (pcDNA3.1-V5-hTEV, Addgene plasmid 65800).

Techniques: Purification, Expressing, Fluorescence, Activation Assay, Incubation, Activity Assay, Cell Culture, Two Tailed Test

Journal: ACS synthetic biology

Article Title: Synthetic circuit-driven expression of heterologous enzymes for disease detection

doi: 10.1021/acssynbio.1c00133

Figure Lengend Snippet: OVCAR8 cells were transduced either with the output virus carrying the TEV protease expression cassette ([0,0]), or the output virus with either of two input viruses ([1,0], [0,1]), or the output virus and both of the input viruses ([1,1]). After two days, cells were fixed to measure the TEV protein output, either by immunofluorescence imaging or flow cytometry analysis via detection of the V5 epitope tag. A) Representative images of TEV abundance (green; SecTEV QSG) in OVCAR8 cells with different input and output configurations. Slides were counterstained with DAPI (blue). Scale bar: 100μm. B) Flow cytometry analysis for V5, with which the TEV protein is tagged, in OVCAR8 cells transduced with different input and output configurations. C) Quantification of the percent of transduced OVCAR8 cells positive for TEV, as measured by anti-V5 signal intensity. Mean ± s.d.; N=3; ordinary one-way ANOVA with Tukey’s correction for multiple comparisons, **** P <0.0001. D, E) Culture medium of circuit-transduced OVCAR8 cells was collected to measure the activity of circuit-outputted TEV in various input and output configurations. Activity was monitored as increase in fluorescence signal over time (D) as measured by the FRET-based reporter and quantified using fluorescence fold change at 120 minutes (E). Mean ± s.d.; N=3; ordinary one-way ANOVA with Tukey’s correction for multiple comparisons, ** P =0.0022 for [1,1] vs. [0,0], ** P =0.0015 for [1,1] vs. [1,0], *P =0.0284 for [1,1] vs. [0,1].

Article Snippet: For the output plasmid, synthetic promoter GAL4BS was placed upstream of transmembrane GFP (hTFR-GFP, Addgene plasmid 45060) or TEV protease (pcDNA3.1-V5-hTEV, Addgene plasmid 65800).

Techniques: Virus, Expressing, Immunofluorescence, Imaging, Flow Cytometry, Transduction, Activity Assay, Fluorescence

Journal: ACS synthetic biology

Article Title: Synthetic circuit-driven expression of heterologous enzymes for disease detection

doi: 10.1021/acssynbio.1c00133

Figure Lengend Snippet: A) OVCAR8 tumor-bearing mice were delivered either the output virus bearing the control cassette alone ([0,0]) or the complete sense-and-respond circuit (SecTEV QSG circuit, [1,1]). One week post viral delivery, the TEV-sensitive in vivo nanosensor was injected i.v., and urine was collected 1.5 hours post injection. In parallel, blood was drawn for the ex vivo readout of TEV activity via the FRET-based reporter. B) Abundance of TEV protease (green; SecTEV QSG) in tumor sections from mice transduced with either the output virus alone ([0,0], left) or the complete sense-and-respond circuit ([1,1], right). Scale bar: 100 μm for upper panels, 20 μm for lower panels. C) Signal-to-noise ratio ([1,1] / [0,0]) of reporter concentration in the urine collected from mice delivered viral vectors defining the [0,0] and [1,1] states. Mean ± s.d.; N=5 mice per group; unpaired two-tailed t-test, ** P =0.0078. D, E) Kinetics (D) and fluorescence fold change (E) of TEV activity in the blood from OVCAR8 tumor-bearing mice, measured ex vivo by fluorescence activation of FRET-paired reporter. Mean ± s.d.; N=5 mice for [0,0] group, N=4 mice for [1,1] group; unpaired two-tailed t-test, ** P =0.0027.

Article Snippet: For the output plasmid, synthetic promoter GAL4BS was placed upstream of transmembrane GFP (hTFR-GFP, Addgene plasmid 45060) or TEV protease (pcDNA3.1-V5-hTEV, Addgene plasmid 65800).

Techniques: Virus, Control, In Vivo, Injection, Ex Vivo, Activity Assay, Transduction, Concentration Assay, Two Tailed Test, Fluorescence, Activation Assay