Additional file 1: of A mathematical model of mechanotransduction reveals how mechanical memory regulates mesenchymal stem cell fate decisions

Supplementary information. This PDF contains supplementary results, references and Figures S1-S3 that are not included in the main text. (PDF 191 kb

Colorimetric and Ultrasensitive Bioassay Based on a Dual-Amplification System Using Aptamer and DNAzyme

Rapid detection of ultralow amount of biomarkers in a biologically complex mixture remains a major challenge. Herein, we report a novel aptamer-based protein detection assay that integrates two signal amplification processes, namely, polymerase-mediated rolling-circle amplification (RCA) and DNA enzyme-catalyzed colorimetric reaction. The target biomarker is captured in a sandwich assay by primary aptamer-functionalized microbeads (MBs) and a secondary aptamer that is connected to a RCA primer/circular template complex. RCA reaction, which amplifies the single biomarker binding events by a factor of hundreds to thousands (the first amplification) produces a long DNA molecule containing multiple DNAzyme units. The peroxidase-like DNAzyme catalyzes the oxidation of 2,2′-azino-bis­(3-ethylbenzothiazoline-6-sulfonic acid) (the second amplification), which generates a blue-green colorimetric signal. This new biosensing platform permits the ultrasensitive, label-free, colorimetric detection of biomarker in real time. Using platelet-derived growth factor B-chain (PDGF-BB) as a model system, we demonstrated that our assay can detect a protein marker specifically in a serum-containing medium, at a concentration as low as 0.2 pg/mL in ∼2 h, which rivals traditional assays such as ELISA. We anticipate this simple methodology for biomarker detection can find utility in point-of-care applications

The Multi-Aptamer does not induce apoptosis or affect cell viability.

<p>(A) Jurkat cells were untreated or treated with SC-Multi-Aptamer, LS-Multi-Aptamer. Treatment with cyclosporine A (CA) served as a control. Apoptosis was assessed at the indicated time points by flow cytometry analysis of annexin V and propidium iodide (PI). (B) To assess potential effects on cell viability or proliferation, Jurkat cells were treated with the indicated compounds. Cell viability was assessed by introduction of XTT reagent. Treatment with 50 μM CA served as a control. Error bars are standard error of the mean (SEM).</p

Facile Supermolecular Aptamer Inhibitors of L-Selectin

<div><p>Multivalent interactions occur frequently in nature, where they mediate high-affinity interactions between cells, proteins, or molecules. Here, we report on a method to generate multivalent aptamers (Multi-Aptamers) that target L-selectin function using rolling circle amplification (RCA). We find that the L-selectin Multi-Aptamers have increased affinity compared to the monovalent aptamer, are specific to L-selectin, and are capable of inhibiting interactions with endogenous ligands. In addition, the Multi-Aptamers efficiently inhibit L-selectin mediated dynamic adhesion in vitro and homing to secondary lymphoid tissues in vivo. Importantly, our method of generating multivalent materials using RCA avoids many of the challenges associated with current multivalent materials in that the Multi-Aptamers are high affinity, easily produced and modified, and biocompatible. We anticipate that the Multi-Aptamers can serve as a platform technology to modulate diverse cellular processes.</p></div

LS-Multi-Aptamer specifically binds to L-selectin on Jurkat cells.

<p>(A) Flow cytometry histograms of Jurkat cells stained with the indicated Multi-Aptamers and controls. (B) Confocal microscope images demonstrate that FITC-labeled LS-Multi-Aptamer (green) effectively binds to Jurkat cells (red). (C) Confocal microscope images demonstrate that cross-linking of multiple Jurkat cells by the LS-Multi-Aptamer can be occasionally found. Scale bar is 5 μm.</p

LS-Multi-Aptamer inhibits dynamic adhesion and homing in vitro and in vivo.

<p>(A) Representative images of Jurkat cells after dynamic adhesion to activated endothelial cells and treatment with the indicated aptamers or Multi-Aptamers. Scale bar is 20 μm. (B) Quantification of Jurkat cells that dynamically adhered to activated endothelial cells following the indicated treatments. * p < 0.05; ** p < 0.01. (C) Quantification of relative recruitment of Jurkat cells to the mesenteric lymph nodes following treatment with the monovalent SC- or LS-aptamer or SC- or LS-Multi-Aptamer, normalized to the respective control. Error bars are SEM.</p

Additional file 1: Figure S1. of Exogenous marker-engineered mesenchymal stem cells detect cancer and metastases in a simple blood assay

Firefly and humanized Gaussia luciferases are substrate-specific and not cross-reactive. hGluc-MSCs, Fluc-tdT-MSCs (Fluc-MSCs), and N-MSCs were seeded in 96-well plate. The firefly luciferase substrate D-luciferin (final concentration = 150 μg/ml) or the humanized Gaussia luciferase substrate CTZ (final concentration = 20 μM) was added, and luciferase activity was measured with a plate reader. Error bar: mean ± standard deviation. Exposure time = 2 s. A.U. arbitrary units, CTZ coelenterazine, Fluc firefly luciferase, hGluc humanized Gaussia luciferase, MSC mesenchymal stem cell, tdT tdTomato red fluorescent protein. (PNG 37 kb

LS-Multi-Aptamer has a higher binding affinity for L-selectin than the monovalent aptamer.

<p>(A) Jurkat cells were incubated with fluorescent monovalent L-selectin aptamer (LS-Aptamer) or the LS-Multi-Aptamer at the indicated concentrations and fluorescence assessed with flow cytometry. (B) Jurkat cells were simultaneously treated with FITC-labeled blocking antibody, DREG56 (100 nM) and increasing concentrations of the indicated reagents. The mean fluorescence for each sample is normalized to the mean fluorescence of the untreated sample labeled with FITC-DREG56.</p

Synthesis of the Multi-Aptamers.

<p>Following a 10 minute RCA reaction, the LS- and SC-Multi-Aptamer DNA products were only generated in the presence of the primer (LS, SC). The negative sample (-) does not contain a primer.</p

DNA sequences used in this study.

<p>DNA sequences used in this study.</p