Journal: bioRxiv

Article Title: 3’ to 5’ translation of linear mRNAs?

doi: 10.64898/2025.12.29.696803

Figure Lengend Snippet: (A) Diagram showing pcDNA3.1(+) ZKSCAN1 MCS as the vector for circular mRNA backward translation. The construct consisted of two backward eGFP fragments (coordinates: 593→1 and 720→594), separated by a TIS-containing sequence. The TIS was flanked by a backward start codon (←GUA) and a backward stop codon (AAU←). (B) Representative fluorescence microscopy images illustrating positive cells after transfection of HEK-293T cells with circular (bw)-eGFP (w/o TIS), hs .circIFITM1-TIS-(bw)-eGFP, hs .circCAPN15-TIS-(bw)-eGFP, and hs .circSCRIB-TIS-(bw)-eGFP. Scale bar, 20μm. Live cells were stained with Mito-Tracker (red, mitochondria). (C) Representative fluorescence microscopy images illustrating HEK-293T cells transfected with the empty vector plasmid, as the negative control. Scale bar, 60μm. HEK-293T cells transfected with the Addgene plasmid #69909 (pcDNA3.1(+) ZKSCAN1 MCS-circ (fw)-eGFP) served as the positive control, with approximately 35%–40% of cells displayed green fluorescence. Scale bar, 40μm. (D) Diagram showing pcDNA3.1(+) as the vector for linear mRNA backward translation. A backward GUA (←GUA) and a backward stop codon (AAU←) were inserted before the TIS. (E) Representative fluorescence microscopy images illustrating positive cells after transfection of HEK-293T cells with linear (bw)-eGFP (w/o TIS), hs .circIFITM1-TIS-(bw)-eGFP, hs .circCAPN15-TIS-(bw)-eGFP, and hs .circSCRIB-TIS-(bw)-eGFP. Scale bar, 20μm. Live cells were stained with Mito-Tracker (red, mitochondria). (F) Representative fluorescence microscopy images illustrating HEK-293T cells transfected with the empty vector plasmid, as the negative control. Scale bar, 60μm. HEK-293T cells transfected with the pcDNA3.1-linear (fw) -eGFP plasmid as the positive control with approximately 85–90% of cells displayed green fluorescence. Scale bar, 60μm. (G) Comparison of linear and circular backward translation effiency in the HEK-293T transfection experiments. The Y axis shows the ratio of positive cells after transfection of HEK-293T cells with circular RNA and linear RNA, respectively. Statistical comparisons between groups 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 vector pcDNA3.1(+) ZKSCAN1 MCS + Sense IRES (Addgene: #69909) was linearized using HindIII and XbaI.

Techniques: Plasmid Preparation, Construct, Sequencing, Fluorescence, Microscopy, Transfection, Staining, Negative Control, Positive Control, Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: 3’ to 5’ translation of linear mRNAs?

doi: 10.64898/2025.12.29.696803

Figure Lengend Snippet: (A) Diagram showing design of the pcDNA3.1(+) vector for bidirectional translation of linear mRNAs. The backward (bw)-eGFP was placed upstream of the TIS, and the forward (fw) mScarlet3 was placed downstream of the TIS. (B) Representative fluorescence microscopy images illustrating HEK-293T cells transfected with the constructed linear bidirectional fluorescence (bw)-eGFP- hs .circIFITM1-TIS-(fw)-mScarlet3. Scale bar, 20 µm. (C) Representative fluorescence microscopy images illustrating HEK-293T cells transfected with the constructed linear bidirectional fluorescence (bw)-eGFP- dm .circSCRIB-TIS-(fw)-mScarlet3. Scale bar, 20 µm. (D) Representative fluorescence microscopy images illustrating HEK-293T cells transfected with the linear constructed bidirectional fluorescence (bw)-eGFP- hs .circCAPN15-TIS-(fw)-mScarlet3. Scale bar, 20 µm. (E) Diagram showing design of the pcDNA3.1(+) ZKSCAN1 MCS vector for bidirectional translation of circular RNAs. The backward (bw)-eGFP was placed upstream of the TIS, and the forward (fw) mScarlet3 was downstream of the TIS. (F) Representative fluorescence microscopy images illustrating HEK-293T cells transfected with the constructed circular bidirectional fluorescent circular (bw)-eGFP- dm .circSCRIB-TIS-(fw)-mScarlet3. Scale bar, 20 µm. (G) Representative fluorescence microscopy images illustrating HEK-293T cells transfected with the constructed circular bidirectional fluorescent (bw)-eGFP- hs .circCAPN15-TIS-(fw)-mScarlet3. Scale bar, 20 µm. (H) Representative fluorescence microscopy images illustrating HEK-293T cells transfected with the constructed circular bidirectional fluorescent (bw)-eGFP- hs .circIFITM1-TIS-(fw)-mScarlet3. Scale bar, 20 µm. (I) Representative fluorescence microscopy images illustrating HEK-293T cells transfected with the constructed circular bidirectional fluorescent (bw)-mScarlet3- hs .circIFITM1-TIS-(fw)-eGFP. Scale bar, 20 µm. Quantifications of the fluorescence positive cells ratio after transfection were shown below each microscopy images panel.

Article Snippet: The vector pcDNA3.1(+) ZKSCAN1 MCS + Sense IRES (Addgene: #69909) was linearized using HindIII and XbaI.

Techniques: Plasmid Preparation, Fluorescence, Microscopy, Transfection, Construct