T800 and T1000 aptamers enforce myelopoiesis in zebrafish.

a General workflow (created with Bio Render). Representative pictures of mpx:GFP (b) and mpeg1:GFP (c) larvae tail and lcr:GFP (d) complete larvae by 48 h post-fertilization (hpf) microinjected with the indicated aptamers. Right panels show quantification of neutrophils (b), macrophages (c) and erythrocyte fluorescence intensity in the white boxed area (d). e Representative images of mpx:GFP larvae transected tails from 1 to 8 h post-wounding (hpw) and quantification of neutrophil migration to the wound as neutrophil number mean±s.e.m. for all larvae. b Data are average of 5 independent experiments. Each dot represents a single larva (Control, n = 315; terc, n = 223; CR7, n = 120; T800, n = 151; T1000mut, n = 224; T1000, n = 219). Mean ± SEM for each group is also shown. Statistical analysis according to ordinary 1way ANOVA followed by Dunnett’s multiple comparison test (95% confidence interval). Scale bar: 250 um. c Data are average of 3 independent experiments. Each dot represents a single larva (Control, n = 97; terc, n = 84; CR7, n = 88; T800, n = 93; T1000mut, n = 92; T1000, n = 102). Mean ± SEM for each group is also shown. Statistical analysis according to ordinary 1way ANOVA followed by Dunnett’s multiple comparison test (95% confidence interval). Scale bar: 250 um. d Data are average of 3 independent experiments. Each dot represents a single larva (Control, n = 72; terc, n = 34; CR7, n = 75; T800, n = 71; T1000mut, n = 46; T1000, n = 75). Mean ± SEM for each group is also shown. n.s., not significant, p > 0.05 according to ordinary 1way ANOVA followed by Dunnett’s multiple comparison test (95% confidence interval). Scale bar: 500 um. e Data are shown as mean ± SEM of 3 independent experiments (Control, n = 30; T1000mut, n = 38; T1000, n = 43). Differences are statistically significant between T1000mut and T1000 groups. Statistical analysis according to mixed-effects analysis followed by Tukey’s multiple comparison test (95% confidence interval). Scale bar: 100 um. Image was created with BioRender.com by co-Authors. Source data are provided as a Source Data file.

T1000 aptamer enforce myelopoiesis by activating gcsf3b tercbs and gcsfa/b expression.

a Luciferase assay using wild type or mutant tercbs of csf3b promoter. Luciferase activity was normalized to Renilla activity and the results shown as fold change relative to control. b, c Representative images and neutrophil quantification of 72 hpf Tg(mpx:GFP) tail larvae. Larvae were microinjected with the indicated aptamer in combination with TALEN targeting the gcsf3b tercbs promoter (b) or gRNAs against csf3a/b genes (c). Data are average of 3 independent experiments. a Each dot represents a biologically independent sample consisting of a pool of 20 larvae (Left: Control, n = 19; terc, n = 16; CR7, n = 10; T800, n = 19; T1000mut, n = 15; T1000, n = 18. Right: Control, n = 13; terc, n = 14; CR7, n = 14; T800, n = 14; T1000mut, n = 14; T1000, n = 14). Bars represent mean ± SEM for each group. n.s., not significant, p > 0.05 according to Kruskal-Wallis followed by Dunn’s multiple comparison test (95% confidence interval). b Each dot represents a single larva (Control, n = 76; T1000mut, n = 84; T1000, n = 109; TALEN, n = 75; TALEN+T1000mut, n = 51; TALEN+T1000, n = 77). Mean ± SEM for each group is also shown. Statistical analysis according to ordinary 1way ANOVA followed by Sidak’s multiple comparison test (95% confidence interval). Scale bar: 200 um. c Each dot represents a single larva (Control, n = 59; T1000mut, n = 66; T1000, n = 81; _gcsf3a+b, n = 56; _gcsf3a+b +T1000mut, n = 59; _gcsf3a+b +T1000, n = 71). Mean ± SEM for each group is also shown. Statistical analysis according to ordinary 1way ANOVA followed by Sidak’s multiple comparison test (95% confidence interval). Scale bar: 200 um. Source data are provided as a Source Data file.

Aptamers physically bind to DNA and to RNApolII.

aterc RNA levels determined by RT-qPCR in 48hpf larvae microinjected with full terc or the indicated aptamers. Gene expression was normalized to rps11 and then to the control sample. b Relative telomerase activity (RTA) measured by qPCR in protein extracts of 48 hpf larvae in the indicated conditions. c RT-qPCR of in vitro DNA-binding assay eluates showing the affinity for the wildtype csf3b promoter related to the csf3b promoter with mutated terc binding site (tercbs), and compared to Luc control. d RT-qPCR of in vivo DNA-binding assay eluates showing the affinity for the CSF2 promoter TERCbs or the SPI 3’UTR TERCbs. e Western blot and quantification of aptamer RNA pulldown eluates using anti-phospho-serine 5 RNA Pol II antibody. a Data are average of 3 biologically independent samples. Each dot represents a pool of 20 larvae. Bars represent mean ± SEM. Statistical analysis according to Kruskal-Wallis followed by uncorrected Dunn’s test (95% confidence interval). b Data are average of 3 independent experiments. Each dot represents a biologically independent sample consisting of a pool of 25 larvae (Control, n = 6; BIBR1532, n = 6; terc, n = 8; CR7, n = 6; T800, n = 9; T1000mut, n = 9; T1000, n = 9). Bars represent mean ± SEM. Statistical analysis according to Kruskal-Wallis followed by uncorrected Dunn’s test (95% confidence interval). c Data are average of 4 independent experiments. Bars represent mean ± SEM. Statistical analysis according to Kruskal-Wallis followed by Dunn’s multiple comparison test (95% confidence interval). d Data are average of 3-4 independent experiments. Bars represent mean ± SEM. Statistical analysis according to Kruskal-Wallis followed by uncorrected Dunn’s test (95% confidence interval). e Data are average of 2 independent experiments. Each dot represents a biologically independent sample consisting of a pool of 25 larvae. Bars represent mean. Images were created with BioRender.com by co-authors. Source data are provided as a Source Data file.

Aptamers rescue neutropenia in zebrafish models of DC and PN.

a Neutrophils quantification at 48 hpf in terc +/- (obtained from mpx:GFP fish outcrossed with terc -/-) and terc -/- (obtained from a terc -/- incross) larvae microinyected with the indicated RNAs. terc -/- larvae were stained with TSA reagent to visualize neutophils. b Representative pictures and neutrophils (dsRed positive cells) quantification at 72 hpf in the tail of lyz:dsRED larvae microinjected with usb1 or std gRNA plus recombinant Cas9 in presence or absence of gcsfb mRNA. The white boxes show the quantified area. c Representative images and neutrophils quantification at 72 hpf in lyz:dsRED larval tail microinjected with usb1 or std gRNA plus recombinant Cas9 and the indicated aptamers. a Each dot represents a single larva (Left: +/+ Control, n = 126; +/- Control, n = 96; +/- terc, n = 48; +/- CR7, n = 52; +/- T800, n = 57; +/- T1000mut, n = 52; +/- T1000, n = 54. Right: +/+ Control, n = 11; -/- Control, n = 16; -/- terc, n = 26; -/- CR7, n = 11; -/- T800, n = 20; -/- T1000mut, n = 24; -/- T1000, n = 25). Mean ± SEM for each group is also shown. Left: Statistical analysis according to ordinary 1way ANOVA followed by Dunnett’s multiple comparison test (95% confidence interval). Right: Statistical analysis according to Kruskal-Wallis followed by Dunn’s multiple comparison test (95% confidence interval). b Data are average of 3 independent experiments. Each dot represents a single larva (Control+_gstd, n = 41; Control+_gusb1, n = 40; gcsfb+_gstd, n = 25; gcsfb+_gusb1, n = 37). Mean ± SEM for each group is also shown. Statistical analysis according to ordinary 1way ANOVA followed by Dunnett’s multiple comparison test (95% confidence interval). Scale bar: 250 um. c Data are average of 3 independent experiments. Each dot represents a single larva (Control+_gstd, n = 42; Control+_gusb1, n = 36; terc+_gstd, n = 46; terc+_gusb1, n = 39; CR7+_gstd, n = 21; CR7+_gusb1, n = 22; T800+_gstd, n = 39; T800+_gusb1, n = 32; T1000mut+_gstd, n = 38; T1000mut+_gusb1, n = 38; T1000+_gstd, n = 42; T1000+_gusb1, n = 39). Mean ± SEM for each group is also shown. Statistical analysis according to ordinary 1way ANOVA followed by Sidak’s multiple comparison test (95% confidence interval). Scale bar: 250 um. Image was created with BioRender.com by co-Authors. Source data are provided as a Source Data file.

Aptamers rescue defective myelopoiesis in mouse and iPSC from DC patients.

a Left graph; comparison of the composition of erythroid and myeloid colonies assayed by colony assays, using whole bone marrow cells from mice of the indicated phenotype. Middle and right graphs; composition of erythroids and myeloids assayed by colony assays, using whole bone marrow cells from mice of the indicated phenotype an incubated with the mouse T1000 aptamer. N = 7 for wt and Terc -/- mice and N = 5 for Tert -/- mice. b Induced-pluripotent stem cell (iPSC) workflow. c Composition of erythroids and myeloids assayed by colony assays, using embryonic bodies (EBs) derived from the indicated iPSC lines treated with different aptamers. d Quantification of the hemogenic progenitors (HEPs) population (CD31 + /CD34 + /CD45-) by flow cytometry at day 15 in EBs. e mRNA levels of SPI1 assayed by RT-qPCR in the different conditions. The expression is normalized to GAPDH and relative to control sample. a Data are average of at least 5 biologically independent samples (wt, n = 7; Tert-/-, n = 5; Terc-/-, n = 7). Bars represent mean ± SEM. Statistical analysis according to 2way ANOVA followed by Dunnett’s multiple comparison test (95% confidence interval). c Data are average of 3 biologically independent samples. Bars represent mean ± SEM. Statistical analysis according to 2way ANOVA followed by Dunnett’s multiple comparison test (95% confidence interval). d Data are average of 3 biologically independent samples represented by dots. Bars represent mean ± SEM for each group. n.s., not significant, p > 0.05 according to Kruskal-Wallis followed by Dunn’s multiple comparison test (95% confidence interval). e Data are average of 3 biologically independent samples represented by dots. Bars represent mean ± SEM. Statistical analysis according to Kruskal-Wallis followed by Dunn’s multiple comparison test (95% confidence interval). Source data are provided as a Source Data file.