Journal: bioRxiv

Article Title: Foliar mRNA spray induces protein synthesis in monocot crop and dicot model plant species

doi: 10.1101/2025.08.07.668951

Figure Lengend Snippet: ( a ) Schematic representation of synthetic mRNA constructs encoding eGFP or citrine, each carrying a C-terminal 3×HA tag. ( b ) Western blot showing in vitro translation products from 5 µg mRNA in wheat germ extract. Controls: Total protein from E. coli BL21 cells overexpressing the respective proteins (100 µg). Detection was performed with primary anti-HA (mouse) and secondary anti-mouse peroxidase (goat) antibodies. Expected molecular weights: eGFP – 30.2 kDa; citrine – 32.7 kDa. Ponceau S staining confirms equal loading. ( c ) Barley protoplasts from 2-week-old seedlings were treated with citrine mRNA via PEG-mediated transfection (with carrier plasmid) or passive incubation. A constitutively citrine-expressing plasmid served as a positive control. CLSM images were taken 15 h post-treatment. Citrine fluorescence was excited at 514 nm (2.6% YFP laser intensity), detected at 508–588 nm. Scale bar: 20 µm.

Article Snippet: StemMACSTM eGFP mRNA (Miltenyi Biotec) was diluted to 100 ng/μl in H 2 O according to the manufacturer’s instructions.

Techniques: Construct, Western Blot, In Vitro, Staining, Transfection, Plasmid Preparation, Incubation, Expressing, Positive Control, Fluorescence

Journal: bioRxiv

Article Title: Foliar mRNA spray induces protein synthesis in monocot crop and dicot model plant species

doi: 10.1101/2025.08.07.668951

Figure Lengend Snippet: The leaves were sprayed with in vitro transcribed GFP mRNA (717 nucleotides) and analyzed for GFP fluorescence 24 hours after treatment. Cross-sections of the leaves were analyzed using CLSM. (A) GFP-positive signals (green) are visible in subepidermal cells (SEC) and in the upper epidermis above the vascular bundles (VB), indicating effective uptake without the need for surfactants. (B) Signal detection in bundle sheath (BS) cells and vascular tissue suggests that the mRNA is accessible to inner tissues. (C) The presence of GFP fluorescence in phloem cells near the xylem implies the potential movement of mRNA or translated protein towards vascular tissues. (D) The mock-treated control shows no GFP signal, confirming the specificity of the observed fluorescence in the treated samples. Scale bars: A = 50 µm; B = 10 µm; C = 10 µm; D = 50 µm

Article Snippet: StemMACSTM eGFP mRNA (Miltenyi Biotec) was diluted to 100 ng/μl in H 2 O according to the manufacturer’s instructions.

Techniques: In Vitro, Fluorescence, Control

Journal: bioRxiv

Article Title: Foliar mRNA spray induces protein synthesis in monocot crop and dicot model plant species

doi: 10.1101/2025.08.07.668951

Figure Lengend Snippet: The adaxial site of barley leaves were sprayed with in vitro transcribed GFP mRNA (717 nucleotides) and analyzed for GFP fluorescence 24 hours after treatment. (A) GFP-positive signals (green) are visible in the adaxial epidermal and stomata cells and cavities. Specificity of the observed signals was confirmed in lambda scanning mode. (B) The mock treated control shows no GFP signal. Scale bars 50 µm.

Article Snippet: StemMACSTM eGFP mRNA (Miltenyi Biotec) was diluted to 100 ng/μl in H 2 O according to the manufacturer’s instructions.

Techniques: In Vitro, Fluorescence, Control

Journal: bioRxiv

Article Title: Foliar mRNA spray induces protein synthesis in monocot crop and dicot model plant species

doi: 10.1101/2025.08.07.668951

Figure Lengend Snippet: Confocal laser scanning microscopy (CLSM) images show cross-sections of wheat leaves 24 hours after the foliar application of in vitro transcribed GFP mRNA. The green fluorescence indicates the presence of GFP (A) shows the GFP signal detected in subepidermal cells (SEC) and stomatal cavities (SC) beneath the upper epidermis. GC = guard cells. (B) GFP fluorescence is observed in vascular tissues, including the phloem and xylem, as well as in adjacent lower epidermal cells. (C) The GFP signal is visible in mesophyll cells (MC), subepidermal cells and cells surrounding the vascular bundle (VB) in the lower leaf area. (D) Mock-treated control showing no GFP signal. Scale bars A-C = 50 µm; D = 100 µm.

Article Snippet: StemMACSTM eGFP mRNA (Miltenyi Biotec) was diluted to 100 ng/μl in H 2 O according to the manufacturer’s instructions.

Techniques: Confocal Laser Scanning Microscopy, In Vitro, Fluorescence, Control

Journal: bioRxiv

Article Title: Foliar mRNA spray induces protein synthesis in monocot crop and dicot model plant species

doi: 10.1101/2025.08.07.668951

Figure Lengend Snippet: Confocal laser scanning microscopy (CLSM) images show cross-sections of maize leaves 24 hours after the foliar application of in-vitro transcribed GFP mRNA. The green fluorescence indicates the presence of GFP signal (A) 24 hours and (B) 48 hours in the vascular tissues, including the phloem and xylem. (C) Mock treated control showing no GFP signal. (VB) vascular bundle; (MC) mesophyll cells. Scale bars 50 µm.

Article Snippet: StemMACSTM eGFP mRNA (Miltenyi Biotec) was diluted to 100 ng/μl in H 2 O according to the manufacturer’s instructions.

Techniques: Confocal Laser Scanning Microscopy, In Vitro, Fluorescence, Control

Journal: bioRxiv

Article Title: Foliar mRNA spray induces protein synthesis in monocot crop and dicot model plant species

doi: 10.1101/2025.08.07.668951

Figure Lengend Snippet: GFP mRNA or water control was delivered to A . thaliana rosette leaves by high pressure spraying technique (HPST). 48h post treatment, three different ribosomal fractions and one supernatant fraction was collected in a sucrose gradient: Heavy Polysomes (HP), Light Polysomes (LP), 80S, 60S and 40S ribosomal fragments (SP), Supernatant (TOP). Color intensities for green, gray and blue follow the depicted sample legend in gray. In each sample (N=3 for each condition), total mRNA level was set to 100 (HP+LP+SP+Top). RNA extracts from each fragment were reverse-transcribed and quantitative PCR was used to assess the level of occupancy of GFP (green) and two housekeeping genes, ACT2 (dark blue) and UBQ10 (blue). in each fraction, the value shows a percentage in total mRNA level. Puromycin treatment dissociated polysomes mRNAs are released to upper fractions. Col0 + GFP refers to HPST of GFP mRNA on Col0, Col0+ water refers to HPST of water on Col0, Puromycin treatment is used to verify that mRNAs. rdr6-11 (Col0 background) is a homozygous deletion of RNA-Dependent-RNA polymerase 6. Error bars indicate Standard error.

Article Snippet: StemMACSTM eGFP mRNA (Miltenyi Biotec) was diluted to 100 ng/μl in H 2 O according to the manufacturer’s instructions.

Techniques: Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Therapy. Nucleic Acids

Article Title: mRNA-based in vivo induction of ETV2 to restore blood flow in a preclinical mouse hindlimb ischemia model

doi: 10.1016/j.omtn.2025.102592

Figure Lengend Snippet: Synthetic mRNA encoding ETV2 is efficiently translated into functional protein in vitro (A) EGFP or ETV2 protein expression at 15 h after mRNA transfection with MessengerMAX. Representative blight filed image and fluorescence image. Scale bars, 75 μm. TF, transfection reagent. (B) After mRNA transfection with MessengerMAX, the cells were collected at 24, 48, or 72 h time point. Total RNA extraction, cDNA synthesis, and qPCR analysis by ΔΔCT method were conducted. For each condition, n = 4; mean ± SD. ∗∗ p < 0.01 by two-way ANOVA with Bonferroni correction. (C) CDH5 protein expression at 24, 48, and 72 h time points after mRNA transfection were analyzed by western Blotting. The number above each gel lane represent the fold-change in intensity relative to control (TF only, 24 h).

Article Snippet: CleanCap EGFP mRNA, CleanCap Fluc mRNA, CleanCap Cre mRNA, and CleanCap Codon-optimized ETV2 mRNA (N1mΨ, RP-HPLC purified) were synthesized at TriLink Biotechnologies (San Diego, CA).

Techniques: Functional Assay, In Vitro, Expressing, Transfection, Fluorescence, RNA Extraction, cDNA Synthesis, Western Blot, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: mRNA-based in vivo induction of ETV2 to restore blood flow in a preclinical mouse hindlimb ischemia model

doi: 10.1016/j.omtn.2025.102592

Figure Lengend Snippet: In vivo biodistribution of the injected ETV2 mRNA with LNP and protein expression in skeletal muscle (A) ETV2 mRNA visualization with RNAscope. Quadriceps were collected 5 h after LNP (0.5 μg/limb) administration intramuscularly. Longitudinal section of the whole quadriceps was analyzed. Scare bars, 3 mm (low-magnification). (B and C) Representative images of (B) ETV2 mRNA visualization with RNAscope and (C) ETV2 protein visualization with IHC. Scale bars, 100 μm (high-magnification).

Article Snippet: CleanCap EGFP mRNA, CleanCap Fluc mRNA, CleanCap Cre mRNA, and CleanCap Codon-optimized ETV2 mRNA (N1mΨ, RP-HPLC purified) were synthesized at TriLink Biotechnologies (San Diego, CA).

Techniques: In Vivo, Injection, Expressing, RNAscope

Journal: Molecular Therapy. Nucleic Acids

Article Title: mRNA-based in vivo induction of ETV2 to restore blood flow in a preclinical mouse hindlimb ischemia model

doi: 10.1016/j.omtn.2025.102592

Figure Lengend Snippet: Profiling the cellular functional distribution of intramuscularly administrated mRNA/LNP (A) Schematic of the Ai6 transgenic mouse LoxP-flanked stop cassette preventing the transcription of ZsGreen. Upon delivery of Cre recombinase via Cre mRNA, the stop cassette is excised, and the cell expresses ZsGreen. (B) Experimental design of the functional delivery assay in vivo . (C) Gating strategy for each cell population: leukocytes, endothelial cells (ECs), and fibro/adipogenic progenitors (FAPs). (D) Flow cytometry scatter plots of ZsGreen expression in each cell population in PBS or Cre mRNA injected Ai6 mice. (E) ZsGreen expression in each cell population ( n = 2 mice). Data are represented as mean ± SD. ∗∗ p < 0.01.

Article Snippet: CleanCap EGFP mRNA, CleanCap Fluc mRNA, CleanCap Cre mRNA, and CleanCap Codon-optimized ETV2 mRNA (N1mΨ, RP-HPLC purified) were synthesized at TriLink Biotechnologies (San Diego, CA).

Techniques: Functional Assay, Transgenic Assay, In Vivo, Flow Cytometry, Expressing, Injection

Journal: Molecular Therapy. Nucleic Acids

Article Title: mRNA-based in vivo induction of ETV2 to restore blood flow in a preclinical mouse hindlimb ischemia model

doi: 10.1016/j.omtn.2025.102592

Figure Lengend Snippet: LNP encapsulating ETV2 mRNA promotes blood flow supply in a hindlimb ischemia model using BALB/c athymic mice (A) Experimental design of HLI study. On day 0 post the HLI surgery, PBS or LNP (3 μg/limb) was administered intramuscularly once a week up to 4 weeks. ETV2 lentivirus was administered intramuscularly on day 0 post HLI surgery. Blood flow was analyzed by LDI by day 35. (B) Representative LDI images on day 0 pre/post HLI surgery and day 14 post HLI surgery. (C) The ratio of blood flow (left injured limb/right normal limb) at all time points for all groups was shown. For each group, n = 6–15; mean ± SE. (D) The ratio of blood flow (left injured limb/right normal limb) at day 14 and day 28 was shown. ∗ p < 0.5 and ∗∗ p < 0.01 by two-way ANOVA with Bonferroni correction.

Article Snippet: CleanCap EGFP mRNA, CleanCap Fluc mRNA, CleanCap Cre mRNA, and CleanCap Codon-optimized ETV2 mRNA (N1mΨ, RP-HPLC purified) were synthesized at TriLink Biotechnologies (San Diego, CA).

Techniques:

Journal: bioRxiv

Article Title: Foliar mRNA spray induces protein synthesis in monocot crop and dicot model plant species

doi: 10.1101/2025.08.07.668951

Figure Lengend Snippet: ( a ) Schematic representation of synthetic mRNA constructs encoding eGFP or citrine, each carrying a C-terminal 3×HA tag. ( b ) Western blot showing in vitro translation products from 5 µg mRNA in wheat germ extract. Controls: Total protein from E. coli BL21 cells overexpressing the respective proteins (100 µg). Detection was performed with primary anti-HA (mouse) and secondary anti-mouse peroxidase (goat) antibodies. Expected molecular weights: eGFP – 30.2 kDa; citrine – 32.7 kDa. Ponceau S staining confirms equal loading. ( c ) Barley protoplasts from 2-week-old seedlings were treated with citrine mRNA via PEG-mediated transfection (with carrier plasmid) or passive incubation. A constitutively citrine-expressing plasmid served as a positive control. CLSM images were taken 15 h post-treatment. Citrine fluorescence was excited at 514 nm (2.6% YFP laser intensity), detected at 508–588 nm. Scale bar: 20 µm.

Article Snippet: Miltenyi StemMACSTM eGFP mRNA was used at a final concentration of 100 ng/μl for spraying.

Techniques: Construct, Western Blot, In Vitro, Staining, Transfection, Plasmid Preparation, Incubation, Expressing, Positive Control, Fluorescence

Journal: bioRxiv

Article Title: Foliar mRNA spray induces protein synthesis in monocot crop and dicot model plant species

doi: 10.1101/2025.08.07.668951

Figure Lengend Snippet: The leaves were sprayed with in vitro transcribed GFP mRNA (717 nucleotides) and analyzed for GFP fluorescence 24 hours after treatment. Cross-sections of the leaves were analyzed using CLSM. (A) GFP-positive signals (green) are visible in subepidermal cells (SEC) and in the upper epidermis above the vascular bundles (VB), indicating effective uptake without the need for surfactants. (B) Signal detection in bundle sheath (BS) cells and vascular tissue suggests that the mRNA is accessible to inner tissues. (C) The presence of GFP fluorescence in phloem cells near the xylem implies the potential movement of mRNA or translated protein towards vascular tissues. (D) The mock-treated control shows no GFP signal, confirming the specificity of the observed fluorescence in the treated samples. Scale bars: A = 50 µm; B = 10 µm; C = 10 µm; D = 50 µm

Article Snippet: Miltenyi StemMACSTM eGFP mRNA was used at a final concentration of 100 ng/μl for spraying.

Techniques: In Vitro, Fluorescence, Control

Journal: bioRxiv

Article Title: Foliar mRNA spray induces protein synthesis in monocot crop and dicot model plant species

doi: 10.1101/2025.08.07.668951

Figure Lengend Snippet: The adaxial site of barley leaves were sprayed with in vitro transcribed GFP mRNA (717 nucleotides) and analyzed for GFP fluorescence 24 hours after treatment. (A) GFP-positive signals (green) are visible in the adaxial epidermal and stomata cells and cavities. Specificity of the observed signals was confirmed in lambda scanning mode. (B) The mock treated control shows no GFP signal. Scale bars 50 µm.

Article Snippet: Miltenyi StemMACSTM eGFP mRNA was used at a final concentration of 100 ng/μl for spraying.

Techniques: In Vitro, Fluorescence, Control

Journal: bioRxiv

Article Title: Foliar mRNA spray induces protein synthesis in monocot crop and dicot model plant species

doi: 10.1101/2025.08.07.668951

Figure Lengend Snippet: Confocal laser scanning microscopy (CLSM) images show cross-sections of wheat leaves 24 hours after the foliar application of in vitro transcribed GFP mRNA. The green fluorescence indicates the presence of GFP (A) shows the GFP signal detected in subepidermal cells (SEC) and stomatal cavities (SC) beneath the upper epidermis. GC = guard cells. (B) GFP fluorescence is observed in vascular tissues, including the phloem and xylem, as well as in adjacent lower epidermal cells. (C) The GFP signal is visible in mesophyll cells (MC), subepidermal cells and cells surrounding the vascular bundle (VB) in the lower leaf area. (D) Mock-treated control showing no GFP signal. Scale bars A-C = 50 µm; D = 100 µm.

Article Snippet: Miltenyi StemMACSTM eGFP mRNA was used at a final concentration of 100 ng/μl for spraying.

Techniques: Confocal Laser Scanning Microscopy, In Vitro, Fluorescence, Control

Journal: bioRxiv

Article Title: Foliar mRNA spray induces protein synthesis in monocot crop and dicot model plant species

doi: 10.1101/2025.08.07.668951

Figure Lengend Snippet: Confocal laser scanning microscopy (CLSM) images show cross-sections of maize leaves 24 hours after the foliar application of in-vitro transcribed GFP mRNA. The green fluorescence indicates the presence of GFP signal (A) 24 hours and (B) 48 hours in the vascular tissues, including the phloem and xylem. (C) Mock treated control showing no GFP signal. (VB) vascular bundle; (MC) mesophyll cells. Scale bars 50 µm.

Article Snippet: Miltenyi StemMACSTM eGFP mRNA was used at a final concentration of 100 ng/μl for spraying.

Techniques: Confocal Laser Scanning Microscopy, In Vitro, Fluorescence, Control

Journal: bioRxiv

Article Title: Foliar mRNA spray induces protein synthesis in monocot crop and dicot model plant species

doi: 10.1101/2025.08.07.668951

Figure Lengend Snippet: GFP mRNA or water control was delivered to A . thaliana rosette leaves by high pressure spraying technique (HPST). 48h post treatment, three different ribosomal fractions and one supernatant fraction was collected in a sucrose gradient: Heavy Polysomes (HP), Light Polysomes (LP), 80S, 60S and 40S ribosomal fragments (SP), Supernatant (TOP). Color intensities for green, gray and blue follow the depicted sample legend in gray. In each sample (N=3 for each condition), total mRNA level was set to 100 (HP+LP+SP+Top). RNA extracts from each fragment were reverse-transcribed and quantitative PCR was used to assess the level of occupancy of GFP (green) and two housekeeping genes, ACT2 (dark blue) and UBQ10 (blue). in each fraction, the value shows a percentage in total mRNA level. Puromycin treatment dissociated polysomes mRNAs are released to upper fractions. Col0 + GFP refers to HPST of GFP mRNA on Col0, Col0+ water refers to HPST of water on Col0, Puromycin treatment is used to verify that mRNAs. rdr6-11 (Col0 background) is a homozygous deletion of RNA-Dependent-RNA polymerase 6. Error bars indicate Standard error.

Article Snippet: Miltenyi StemMACSTM eGFP mRNA was used at a final concentration of 100 ng/μl for spraying.

Techniques: Control, Reverse Transcription, Real-time Polymerase Chain Reaction