Journal: Nucleic Acids Research

Article Title: Engineering polymeric RNA scaffolds as programmable combinatorial innate immune agonists

doi: 10.1093/nar/gkag328

Figure Lengend Snippet: PolyRNAs of varying structures and sequences activate multiple PRRs. ( A ) Proposed structural features of polyRNA for PRR-specific recognition and activation. A dumbbell-shaped DNA template is processively transcribed by T7 RNA polymerase to generate 5′ triphosphate-containing polyRNA structures comprising repeat units of dsRNA and ssRNA regions. Panel of polyRNAs screened for activation of specific PRRs. All polyRNAs in the panel have a 25 bp dsRNA stem in each repeat, with varying ssRNA loop and connecting region lengths and sequences as indicated in the schematics. GUU labels indicate GU-rich ssRNA sequences. ( C, D ) Co-transcriptional structure prediction of monomeric units ( C ) and oligomeric RNAs ( D ) by KineFold . Pseudoknots are visualized as coloured single-stranded regions connected by two straight lines as predicted by KineFold. Structure prediction images were created with KineFold and polished using Adobe Photoshop. ( E ) In vitro activation of PRRs by polyRNAs transfected with Mirus TransIT-X2 (Mirus) in HEK-Blue hTLR3, hTLR7, and Null1 cells at 2 μg/ml. HEK-Blue Null1 is the parental cell line of HEK-Blue TLR cell lines, with baseline PRR expression levels. ( F ) In vitro RIG-I activation by polyRNAs transfected by Lipofectamine 3000 (Lipo) in HEK-Lucia RIG-I cells at 0.5 μg/ml. ( G ) In vitro IRF activation of IRF in RAW-Dual cells by polyRNAs transfected by Lipofectamine at 0.5 μg/ml. For panels (E)–(G), established agonist benchmarks were included for each PRR reporter cell line: high molecular weight poly(I:C) for TLR3, Null1, and RAW-Dual, R848 for TLR7, and 3p-hpRNA for RIG-I. The data represent the mean ± standard deviation of n = 3 technical replicates. Data were analysed by one-way Analysis of Variance (ANOVA) with Šidak’s multiple comparisons test. Ns, no significant difference between bracketed groups. **/***/**** denotes significance between bracketed groups ( P <.01/.001/.0001). Figure and were created in BioRender. Yang, Y. (2026) https://BioRender.com/gd4yhbl .

Article Snippet: As we observed that activation levels by polyRNAs in different reporter cell lines are influenced by the transfection reagent, we used either the commercial lipid-based Lipofectamine 3000 or polymer-based Mirus TransIT-X2 transfection reagent, as optimized for each cell line.

Techniques: Activation Assay, In Vitro, Transfection, Expressing, High Molecular Weight, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Engineering polymeric RNA scaffolds as programmable combinatorial innate immune agonists

doi: 10.1093/nar/gkag328

Figure Lengend Snippet: LNP formulation can be optimized for polyRNAs of varying lengths. ( A ) Structures of polyRNAs used for LNP formulation screening. ( B ) Size distributions of polyRNAs, as analysed by agarose gel electrophoresis. The average length of each polyRNA was determined using ImageJ. T3, T52, T43, and T51 polyRNAs comprise hairpin repeat motifs, while T19 polyRNA comprises nonhairpin, predominantly single-stranded repeats. ( C, D ) Activation of the IRF pathway by polyRNAs delivered by Lipofectamine 3000 (Lipo) and LNPs in RAW-Dual reporter cells, shown as absolute values ( C ) and as percentages of Lipofectamine/polyRNA IRF activation ( D ). ( E, F ) Activation of the NF-κB pathway by polyRNAs delivered by Lipofectamine 3000 (Lipo) and MC3-based LNPs in RAW-Dual reporter cells, shown as absolute values ( E ) and as percentages of Lipofectamine/polyRNA NF-κB activation ( F ). All dosages of polyRNA and the poly(I:C) benchmark were 0.5 µg/ml. MC3 lipid from MedChemExpress was used for LNP synthesis in this figure. The data represent the mean ± standard deviation of n = 3 technical replicates. Data were analysed by one-way ANOVA with Šidak’s multiple comparisons test. ns, no significant difference between bracketed groups. */**/***/**** denotes significance between bracketed groups ( P <.05/.01/.001/.0001). Figure was created in BioRender. Yang, Y. (2026) https://BioRender.com/m44nwii .

Article Snippet: As we observed that activation levels by polyRNAs in different reporter cell lines are influenced by the transfection reagent, we used either the commercial lipid-based Lipofectamine 3000 or polymer-based Mirus TransIT-X2 transfection reagent, as optimized for each cell line.

Techniques: Formulation, Agarose Gel Electrophoresis, Activation Assay, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Engineering polymeric RNA scaffolds as programmable combinatorial innate immune agonists

doi: 10.1093/nar/gkag328

Figure Lengend Snippet: Assembly of CpG-DNA motifs onto polyRNA scaffolds induces activation of multiple PRRs in vitro . ( A ) Structure of a CpG-DNA comb, comprising a region complementary to a polyRNA repeat sequence, a linker, and a CpG-DNA sequence. Agarose gel electrophoresis of T52 polyRNA and DNA comb-patterned polyRNA indicates successful hybridization. T52 polyRNA comprises hairpin repeat motifs and averages 1000 bases in length. Complementary DNA comb: D52. The D52 DNA comb was hybridized to T52 polyRNA at a DNA comb/polyRNA repeat molar ratio of 1:3. ( C, D ) In vitro activation of the IRF ( C ) and NF-κB ( D ) pathways by T19 polyRNA and its CpG-DNA hybrid, transfected by Lipofectamine 3000 (Lipo) in RAW-Dual reporter cells, compared to the PRR agonist benchmarks poly(I:C) and 5′ triphosphate hairpin RNA (3p-hpRNA). A CpG-DNA comb only quantitative control was dosed at the equivalent amount used for hybridizing to polyRNA. T19 polyRNA comprises nonhairpin repeat motifs and averages 7000 bases in length. The D19-CpG comb is a DNA oligo mTLR9 agonist with a complementary region to T19 repeats and was hybridized to T19 polyRNA at a DNA comb/polyRNA repeat molar ratio of 1:4. ( E, F ) In vitro activation of PRRs by T52 polyRNA and its CpG-DNA hybrid, transfected with D-Lin-MC3-DMA (MC3)-based LNPs in RAW-Dual ( E ) and HEK-Blue mTLR9 reporter cells ( F ). D52-CpG is a DNA oligo mTLR9 agonist with a complementary region to T52 repeats. D52.mismatch-CpG is a mismatch comb control that does not contain any region complementary to T52 repeats. CpG-DNA combs were hybridized to T52 polyRNA at a DNA comb/polyRNA repeat molar ratio of 1:3. All dosages of polyRNAs, polyRNA/CpG-DNA (dosing based on polyRNA mass), and benchmark PRR agonists were 0.5 µg/ml. All CpG DNA comb-only samples were dosed at levels equivalent to the amounts hybridized to their corresponding base polyRNA scaffolds. The data represent the mean ± standard deviation of n = 3 technical replicates ( C–F ). Data were analysed by one-way ANOVA with Šidak’s multiple comparisons test. ns, no significant difference between bracketed groups. **/***/**** denotes significance between bracketed groups ( P <.01/.001/.0001). Figure and were created in BioRender. Yang, Y. (2026) https://BioRender.com/99l41mi .

Article Snippet: As we observed that activation levels by polyRNAs in different reporter cell lines are influenced by the transfection reagent, we used either the commercial lipid-based Lipofectamine 3000 or polymer-based Mirus TransIT-X2 transfection reagent, as optimized for each cell line.

Techniques: Activation Assay, In Vitro, Sequencing, Agarose Gel Electrophoresis, Hybridization, Transfection, Control, Standard Deviation