Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: In silico design and validation of high-affinity RNA aptamers targeting epithelial cellular adhesion molecule dimers

doi: 10.1073/pnas.1913242117

Figure Lengend Snippet: EP23 Structure and Mg2+. (A) The Mfold-predicted secondary structure of EP23. (B) The 3D folded structure of EP23 determined from MD simulations, with the 4 base pairs predicted from Mfold highlighted in blue. (C) Base pair dynamics of the Mfold predicted secondary structure. The terminal base pair (blue) changed conformations slightly from a Watson-Crick to a Hoogsteen-like configuration. (D) Mg2+ ions binding nucleotides 8 (top) and 18 (bottom) over the course of the MD simulations. Two long-lived bound ions are circled in red. (E) Mg2+ ion contact ratio mapped onto the EP23 aptamer surface. The two bound Mg2+ ions correspond to the long-lived bound ions in D.

Article Snippet: The original EpCAM aptamer EP23 and the mutant aptamers A5U and G15U were synthesized by Suzhou Genepharma (Suzhou, China), followed by HPLC purification.

Techniques: Binding Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: In silico design and validation of high-affinity RNA aptamers targeting epithelial cellular adhesion molecule dimers

doi: 10.1073/pnas.1913242117

Figure Lengend Snippet: EP23-EpCAM monomer binding results. (A) EpCAM monomer showing the N-terminal domain in green, TY loop in blue, and C-terminal domain in magenta. (B) EpCAM TY loop fluctuations observed in control EpCAM explicit solvent MD simulations. The residues used for contact analysis are shown as spheres in green (TY loop) and brown (core EpCAM). (C) EpCAM mapped contacts from the top 200 Dot2.0 docking conformations, showing the strong preference for TY loop binding conformations. (D, Top) Ten docking conformations predicted by Dot2.0, all targeting the TY loop region at the dimer interface and the membrane side of the protein. The RNA backbone is shown, colored by energy from strong (red) to weak (blue). (E) EpCAM dimer crystal structure, showing dimerization using the TY loop.

Article Snippet: The original EpCAM aptamer EP23 and the mutant aptamers A5U and G15U were synthesized by Suzhou Genepharma (Suzhou, China), followed by HPLC purification.

Techniques: Binding Assay, Control, Solvent, Membrane

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: In silico design and validation of high-affinity RNA aptamers targeting epithelial cellular adhesion molecule dimers

doi: 10.1073/pnas.1913242117

Figure Lengend Snippet: EP23 binding EpCAM dimer. (A) EpCAM dimer, showing dimerization along the TY loop (blue) and exposure of the N and C-terminal domains (green and magenta, respectively). (B) Aptamer EP23 contacts mapped onto the EpCAM dimer from the top 200 Dot2.0 docking conformations. For the dimer system, docked conformations are spread across the protein, but show higher population for the exposed C-terminal domain. (C, Top) 10 Dot2.0 docking conformations for the EpCAM dimer, colored by energy from strong (red) to weak (blue). EpCAM dimer is shown in a surface representation and colored by protein monomer: cyan and gray. (D) Four unique docking conformations from the top 10 docking conformations selected for further binding assessment. EpCAM dimer is shown in a surface representation and colored by protein monomer: cyan and gray, while the EP23 aptamer is illustrated as a cartoon in magenta, with bound Mg2+ ions shown as pink spheres. (E) The work required to pull the EP23 conformation off of EpCAM dimer, with larger work showing stronger binding. The orange line is the median, boxes extend form lower to upper quartiles, whiskers show range of nonoutlier data. (F) Jarzynski equality averages computed from pulling EP23 off of the EpCAM dimer. Values shown are the mean, SE bars are shown but within the data points. (G) The root-mean-square deviation (RMSD) of atomic positions for the selected EP23 binding conformations + EpCAM dimer configurations across the MD simulations. Note: 02_1, 02_2, and 02_3 are 3 replicas of 500-ns MD extension starting from the 500-ns structure of run 02.

Article Snippet: The original EpCAM aptamer EP23 and the mutant aptamers A5U and G15U were synthesized by Suzhou Genepharma (Suzhou, China), followed by HPLC purification.

Techniques: Binding Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: In silico design and validation of high-affinity RNA aptamers targeting epithelial cellular adhesion molecule dimers

doi: 10.1073/pnas.1913242117

Figure Lengend Snippet: EP23 mutation binding affinity. (A) Comparison of Mfold-predicted secondary structures for the original EP23 sequence, the A5U mutation, and the G15U mutation. (B) The change in binding affinity for two mutations, both as predicted by FEP simulations and determined by ITC experiments. Error bars represent 95% CIs. Starred values (*) indicate P < 0.05, and double-starred (**) experimental values indicate P < 0.0001 for a one-sided t test. (C) Isothermal titration calorimetric analysis of the interaction of the 50-μM aptamer solutions from EP23, A5U, or G15U, with EpCAM protein (5 μM) in ITC buffer at 25 °C. (Top and Bottom) Raw data and binding isotherm obtained over a series of injections of aptamer into EpCAM protein. (Top) Differential power (μcal/sec) versus time is presented in the form of integrated heat values. The data were fitted using a one binding site model. BSA control data are presented in SI Appendix, Fig. S2. (D) Structural comparison of bound EP23 (blue cartoon) with the A5U mutation (orange) and the G15U mutation (green). Bound Mg2+ ions shown as pink spheres; EpCAM dimer is shown in a surface representation and colored cyan or gray for different monomers.

Article Snippet: The original EpCAM aptamer EP23 and the mutant aptamers A5U and G15U were synthesized by Suzhou Genepharma (Suzhou, China), followed by HPLC purification.

Techniques: Mutagenesis, Binding Assay, Comparison, Sequencing, Titration, Control