cdna sequencing  (Novogene)

Journal: Nature Communications

Article Title: A reversible allosteric inhibitor of GlyT2 for neuropathic pain without on-target side effects

doi: 10.1038/s41467-026-69616-5

Figure Lengend Snippet: Chemical structure of ( a ) ORG25543 and ( b ) RPIGLYT2-82. c Glycine dose-responses in the presence of different concentrations of RPI-GLYT2-82 showing non-competitive inhibition. A Brown-Forsythe ANOVA test with Dunnett’s T3 multiple comparisons determined there is no statistically significant difference in the glycine EC 50 across all four conditions ( p = 0.49, n = 5 per condition). There is a significant reduction in I/I 300 between 0 nM RPI-GLYT2-82 and 600 nM ( p < 0.0001, n = 5) and 1.8 µM RPI-GLYT2−82 ( p < 0.0001, n = 5), determined by an ordinary one-way ANOVA with Dunnett’s multiple comparisons. There is no significant reduction in I/I 300 between 0 nM and 200 nM RPI-GLYT2-82 ( p = 0.07, n = 5). d RPI-GLYT2-82 concentration-dependent inhibition of glycine transport. The difference between hGlyT2 WT ( n = 6) and hGlyT2 Δ185 ( n = 5) is not significant ( p = 0.45), determined by a two-tailed unpaired T-test. e ORG25543 concentration-dependent inhibition of glycine transport. The difference between hGlyT2 WT ( n = 6) and hGlyT2 Δ185 ( n = 5) is not statistically significantly ( p = 0.60), determined by a two-tailed unpaired T-test. f RPI-GLYT2-82 washes out within 5-min (WT not fitted due to full reversal before the first time point) ( n = 5 for both conditions). g ORG25543 has prolon g ed inhibitory effects with minimal restoration of glycine-induced currents ( n = 5 for both conditions). h RPI-GLYT2-82 inhibits GlyT2 ( n = 5) with minimal effect on GlyT1 ( n = 5) ( p < 0.0001) determined by a two-tailed unpaired T-test. i The RPI-GLYT2-82 IC 50 at 20 mM Na + (IC 50 = 159 nM (95% CI: 81 to 238 nM), n = 5) is significantly lower than 100 mM Na + (IC 50 = 279 nM (95% CI: 182 to 376 nM), n = 5) ( p = 0.04). j The ORG25543 IC 50 at 20 mM Na + (IC 50 = 18 nM (95% CI: 6.8 to 29 nM), n = 5) is not significantly different to 100 mM Na + (IC 50 = 17.6 nM (95% CI: 5.5 to 30 nM), n = 5) ( p > 0.9999). Data in i , j analysed by a two-way ANOVA with Tukey’s multiple comparisons. k Normalised glycine-dependent transport currents showing comparable transport by hGlyT2 WT ( n = 5) and hGlyT2 Δ185 ( n = 5). ( p = 0.78), determined by a two-tailed unpaired T-test. n indicates biological replicates. Error bars represent ± SD from the mean. Source data are provided as a Source Data file.

Article Snippet: The human GlyT2 complementary DNA sequence was codon optimised and synthetised by Azenta for expression in mammalian cells.

Techniques: Inhibition, Concentration Assay, Two Tailed Test

Journal: Nature Communications

Article Title: A reversible allosteric inhibitor of GlyT2 for neuropathic pain without on-target side effects

doi: 10.1038/s41467-026-69616-5

Figure Lengend Snippet: Cryo-EM density maps of hGlyT2 Δ185 in ( a ) substrate-free state (purple map contour level = 0.04 in ChimeraX), bound to b substrate glycine (pink map contour level = 0.046), c ORG25543 (green map contour level = 0.055 in ChimeraX), and d RPI-GLYT2-82 (blue map contour level = 0.038 in ChimeraX). Lipid densities shown in light yellow, densities corresponding to sterols built into the models shown in dark yellow. e-h Surface representation of substrate-free, substrate-bound, and inhibitor-bound structures of hGlyT2 Δ185 viewed parallel to the membrane. e Slice view of hGlyT2 Δ185 in substrate-free form. Closed extracellular vestibule around W215 (blue) and open intracellular pathway are displayed. f Slice view of hGlyT2 Δ185 showing glycine binding pocket (cyan, compound density shown at map contour level = 0.046 in ChimeraX) g Slice view of hGlyT2 Δ185 showing ORG25543 binding pocket (orange, compound density shown at map contour level = 0.055 in ChimeraX). h Slice view of hGlyT2 Δ185 showing RPI-GLYT2-82 binding pocket (dark purple, compound density shown at map contour level = 0.038 in ChimeraX). Residues W215 (TM1, blue), R216 (TM1, blue), P543 (EL4, yellow), and D633 (TM10, orange) are shown as sticks in e-h ; TM6 shown in green in f .

Article Snippet: The human GlyT2 complementary DNA sequence was codon optimised and synthetised by Azenta for expression in mammalian cells.

Techniques: Cryo-EM Sample Prep, Membrane, Binding Assay

Journal: Nature Communications

Article Title: A reversible allosteric inhibitor of GlyT2 for neuropathic pain without on-target side effects

doi: 10.1038/s41467-026-69616-5

Figure Lengend Snippet: a Cartoon representation of substrate-free hGlyT2 Δ185 , glycine-bound hGlyT2 Δ185 , ORG25543 -bound hGlyT2 Δ185 , and RPI-GLYT2-82-bound hGlyT2 Δ185 . b Upper panel is a magnified view of collapsed Na1 site, usual Na1 site-forming residues shown with corresponding densities (contour level = 0.030 in ChimeraX). Lower panel is of Cl – (green) modelled in substrate-free hGlyT2 Δ185 with coordinating residues (mean coordination distance of 3.1 Å ± 0.4) and densities shown (contour level = 0.030 in ChimeraX). c Upper panel is a magnified view of Na + (purple) modelled into Na2 with coordinating residues (mean coordination distance of 2.9 Å ± 0.4) with densities (contour level = 0.046 in ChimeraX). Lower panel is a magnified view of Cl – (green) modelled in glycine-bound hGlyT2 Δ185 with coordinating residues (mean coordination distance of 3.2 Å ± 0.5) and densities (contour level = 0.046 in ChimeraX). d Upper panel is a magnified view of Na + (purple) modelled into the density at Na1 with coordination residues (contour level = 0.050 in ChimeraX). ORG25543 shown in orange and water molecules shown in red (with corresponding densities). Lower panel is a magnified view of Cl – (green) modelled in ORG25543 -bound hGlyT2 Δ185 with coordinating residues (mean coordination distance of 2.8 Å ± 0.3) and densities shown (contour level = 0.050 in ChimeraX). e Cartoon representation of RPI-GLYT2-82-bound hGlyT2 Δ185 . Upper panel is a magnified view of Na + (purple) modelled into the density at Na1 with coordination residues (contour level = 0.045 in ChimeraX). RPI-GLYT2-82 is in magenta and water molecules in red (with corresponding densities). Lower panel shows Cl – (green) modelled in RPI-GLYT2-82-bound hGlyT2 Δ185 with coordinating residues (mean coordination distance of 2.9 Å ± 0.5) and densities (contour level = 0.045 in ChimeraX).

Article Snippet: The human GlyT2 complementary DNA sequence was codon optimised and synthetised by Azenta for expression in mammalian cells.

Techniques:

Journal: Nature Communications

Article Title: A reversible allosteric inhibitor of GlyT2 for neuropathic pain without on-target side effects

doi: 10.1038/s41467-026-69616-5

Figure Lengend Snippet: a Cryo-EM density of glycine (contour level = 0.042 in ChimeraX). b Glycine (cyan) bound to the central site of hGlyT2. Interacting residues shown in sticks, H-bonds and ionic interactions shown as dashed grey lines. Na + modelled into Na2 shown as a green circle, modelled Cl – shown as a purple circle, and modelled water molecule shown as a red circle. c Glycine binds in distinct poses in GlyT2 and GlyT1. Glycine-bound hGlyT2 (pink) overlayed with glycine-bound GlyT1 (PDB ID 8WFI, grey). Interacting residues shown as sticks (hGlyT2 = pink, hGlyT1 = black). d Cryo-EM density of ORG25543 (contour level = 0.055 in ChimeraX). e Cryo-EM density of RPI-GLYT2-82 (contour level = 0.04 in ChimeraX). f ORG25543 (orange) bound to hGlyT2 Δ185 (green). Interacting residues shown as sticks and H-bonds and ionic interactions shown as grey dashed lines. N213 is likely involved in a weak hydrogen bond interaction with the polarised C–H of the methyl group attached to positively charged ammonium nitrogen of R4 substituent. g RPI-GLYT2-82 (purple) bound to hGlyT2 Δ185 (blue). Interacting residues shown as sticks, and H-bonds and ionic interactions shown as grey dashed lines. h , i Chemical structure of h ORG25543 and i RPI-GLYT2-82 with key chemical substituents R1 (blue), R2 (orange), R3 (green), and R4 (yellow) outlined. j ORG25543 concentration-dependant inhibition of glycine transport by hGlyT2 WT (black) ( n = 6), hGlyT2 W215F (red) ( n = 5) and hGlyT2 D633E (blue) ( n = 5). k Reversibility of ORG25543 inhibition of glycine transport currents mediated by hGlyT2 WT ( n = 5), hGlyT2 W215F ( n = 5) and hGlyT2 D633E ( n = 5). l RPI-GLYT2-82 dose-response curves of hGlyT2 WT ( n = 5), hGlyT2 W215F ( n = 6) and hGlyT2 D633E ( n = 5). The IC 50 values for hGlyT2 W215F and hGlyT2 D633E are greater than the maximal tested dose ( > 10 µM). m RPI-GLYT2-82 is a reversible inhibitor of glycine transport by hGlyT2 WT ( n = 5), hGlyT2 W215F ( n = 5), and hGlyT2 D633E ( n = 5). n refers to biological replicates with error bars representing ± SD from the mean. Source data are provided as a Source Data file.

Article Snippet: The human GlyT2 complementary DNA sequence was codon optimised and synthetised by Azenta for expression in mammalian cells.

Techniques: Cryo-EM Sample Prep, Concentration Assay, Inhibition

Journal: Nature Communications

Article Title: A reversible allosteric inhibitor of GlyT2 for neuropathic pain without on-target side effects

doi: 10.1038/s41467-026-69616-5

Figure Lengend Snippet: a RPI-GLYT2-82 reduced response rate to von Frey stimulus in chronic constriction injury (CCI) mice. Two-way ANOVA with Dunnett’s multiple comparison post-hoc test comparing vehicle and every other group showed significant effects of time ( p < 0.001; F (5, 149) = 27.3) and treatment ( p < 0.001; F (3, 28) = 48.3), and an interaction effect ( p < 0.001; F (21, 196) = 6.30). A 50 mg/kg (i.p.) dose of RPI-GLYT2-82 demonstrated efficacy against mechanical allodynia, significant at 2 and 3 hours post-injection compared to vehicle ( p = 0.003, p < 0.001, respectively) A 100 mg/kg dose of RPI-GLYT2-82 showed a similar trend at 2 and 3 hours ( p = 0.001, p < 0.001, respectively). b RPI-GLYT2-82 reduced response rate to acetone stimulus in CCI mice. Two-way ANOVA with Dunnett’s multiple comparison post-hoc test was conducted between CCI model mice administered vehicle and every other group showing significant main effects of time ( p < 0.0001; F (4, 93) = 25.8) and treatment ( p < 0.0001; F (3, 26) = 21.8, and an interaction effect ( p < 0.0001; F (11, 93) = 4.4). Both 50 and 100 mg/kg (i.p.) significantly reduced cold allodynia between 0.5 and 6-hours postinjection compared to vehicle ( p < 0.05-0.001). Gabapentin was also significant at 1-6 hours ( p < 0.05-0.001). c In PSNL mice, RPI-GLYT2-82 reduced von Frey response rates. Two-way ANOVA showed significant main effects of time ( p < 0.001; F (4, 46) = 12.7) and treatment ( p < 0.001; F (2, 13) = 20.8), and an interaction effect ( p < 0.001; F (14, 91) = 5.86). A 50 mg/kg (i.p.) dose reduced mechanical allodynia, significant at 90 minutes and 2-hours post-injection compared to vehicle ( p = 0.02, p = 0.03, respectively). d – f No deficits were observed at 50 mg/kg RPI-GLYT2-82. At 250 mg/kg, RPI-GLYT2-82 caused transient reductions in d rotarod performance, e grip strength (at 30 minutes but fully recovered by 60 minutes), and f sedation. g – i RPI-GLYT2−82 (50 mg/kg and 250 mg/kg) examined against positive morphine control (10 mg/kg) and vehicle (saline). Activity of compounds using whole-body plethysmography, measuring ( g ) respiratory frequency, h min volume, and i tidal volume. Two-way ANOVA with Dunnett’s multiple comparison post-hoc test conducted between mice administered saline and every other group. j RPI-GLYT2-82 did not increase preference in a conditioned place preference paradigm. One-way ANOVA with Dunnett’s multiple comparison post-hoc test conducted between mice administered vehicle and every other group (F (3, 25) = 3.03; p = 0.0482). RPI-GLYT2-82 50 mg/kg ( n = 8, p = 0.78) and 150 mg/kg ( n = 8, p = 0.15) compared to positive 10 mg/kg morphine control ( n = 6, p = 0.0287) and vehicle (5% solutol, 95% PBS) ( n = 7). a – i Data shown as mean ± SEM. J Individual replicates shown with error bars representing ± SEM from the mean. Significance is denoted as: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. n = biological replicates. Source data are provided as a Source Data file.

Article Snippet: The human GlyT2 complementary DNA sequence was codon optimised and synthetised by Azenta for expression in mammalian cells.

Techniques: Comparison, Injection, Control, Saline, Activity Assay, Conditioned Place Preference

Journal: Nature Communications

Article Title: A reversible allosteric inhibitor of GlyT2 for neuropathic pain without on-target side effects

doi: 10.1038/s41467-026-69616-5

Figure Lengend Snippet: Schematic showing conformational differences between inhibitor-bound outward-open, glycine-bound inward-occluded and substrate-free inward-open states of GlyT2. ORG25543 (orange) and RPI-GLYT2-82 (magenta) lock GlyT2 in an outward-open conformation. Key binding site residues stabilizing the compounds, L211, W215, R216, P543, and D633, shown as sticks. The slow dissociation rate of ORG25543 prohibits the transporter cycling to an inward-open state. RPI-GLYT2-82 binding to GlyT2 is reversible allowing the transporter to sample other conformational states after release. Glycine is bound to GlyT2 in the inward-occluded state, S479 forming a key interaction to stabilize glycine in the central pocket. In the substrate-free inward-open state, the extracellular gate formed by R216 and D633 closes access to the extracellular side and W215 occupies the allosteric site. Cl – (green circle) is bound in all states, Na + (purple circle) in Na1 is bound only in the inhibitor-bound states, and Na + in Na2 is observed only in the substrate-bound state. Transmembrane helices TM1, TM6, and TM10 shown in blue, green, and orange, respectively, and EL4 is shown in yellow.

Article Snippet: The human GlyT2 complementary DNA sequence was codon optimised and synthetised by Azenta for expression in mammalian cells.

Techniques: Binding Assay

Journal: ACS Omega

Article Title: Aptamer-Driven Nanozyme-SERS Bifunctional Sensors with Enrichment Separation Effect for Ultrasensitive Detection of Liver-Fibrosis-Related Biomarkers

doi: 10.1021/acsomega.5c12530

Figure Lengend Snippet: Schematic characterization of Fe 3 O 4 @cDNA-H 1 @Au@Pt NPs. (A) SEM image of a single Fe 3 O 4 @cDNA-H 1 @Au@Pt NP. (B) SEM image of multiple Fe 3 O 4 @cDNA-H 1 @Au@Pt NPs. (C) Particle size distribution of Fe 3 O 4 @cDNA-H 1 @Au@Pt NPs. (D) Shell thickness distribution of Fe 3 O 4 @cDNA-H 1 @Au@Pt NPs. (E) EDX spectrum of Fe 3 O 4 @cDNA-H 1 @Au@Pt NPs. (F) Comparison of SERS spectra between DTNB-labeled Fe 3 O 4 @cDNA-H 1 @Au@Pt NPs and pure DTNB.

Article Snippet: Sstreptavidin-modified Fe 3 O 4 nanoparticles (400 nm) and nucleotides mentioned in , including miR-34a, single-base mismatch sequence (MT1), triple-base mismatch sequence (MT3), random sequence, nucleic acid aptamer sequence (cDNA), and nucleic acid aptamer complementary sequence (H 1 ), were all procured from Sangon Biotech (Shanghai) Co., Ltd. (China).

Techniques: Comparison, Labeling

Journal: ACS Omega

Article Title: Aptamer-Driven Nanozyme-SERS Bifunctional Sensors with Enrichment Separation Effect for Ultrasensitive Detection of Liver-Fibrosis-Related Biomarkers

doi: 10.1021/acsomega.5c12530

Figure Lengend Snippet: (A) Fe 3 O 4 @cDNA-H 1 @Au@Pt NPs under an applied external magnetic field and without an external magnetic field. (B) UV–visible absorption spectra of Fe 3 O 4 alone, Au@Pt NPs alone, and Fe 3 O 4 @cDNA-H 1 @Au@Pt NPs in the presence of TMB and H 2 O 2 . The inset shows a photograph. (C) Michaelis–Menten curve of the nanozyme and (D) Lineweaver–Burk plot.

Article Snippet: Sstreptavidin-modified Fe 3 O 4 nanoparticles (400 nm) and nucleotides mentioned in , including miR-34a, single-base mismatch sequence (MT1), triple-base mismatch sequence (MT3), random sequence, nucleic acid aptamer sequence (cDNA), and nucleic acid aptamer complementary sequence (H 1 ), were all procured from Sangon Biotech (Shanghai) Co., Ltd. (China).

Techniques: