Cells with nuclear TDP-43 also displayed nuclear distribution of Apt-1 (Fig. Lett. Ponti, R. D., Armaos, A., Vandelli, A. In the present work, we describe how we stretched the catRAPID algorithm to design short RNA aptamers, by flanking it with a whole pipeline of computational tools. MD calculations were carried out at 300K in explicit solvent. The mean number of localizations per aggregate (Fig. Single-molecule FRET studies on alpha-synuclein oligomerization of Parkinsons disease genetically related mutants. Cells 3D images were reconstructed from the Z-stacks. These authors contributed equally: Elsa Zacco, Owen Kantelberg. Nucleic Acids Res. 3a), as reported in the NMR model, while nApt-1 interacts with amino acids 140143,165,186191, 262263, and 267 (Fig. Disco. We exploited Apt-1 to track TDP-43 aggregation (Fig. : Conceptualization, data analysis, funding acquisition, supervision, and writing. https://doi.org/10.1038/s41467-022-30944-x, DOI: https://doi.org/10.1038/s41467-022-30944-x. Amyotrophic lateral sclerosis: moving towards a new classification system. Nature Communications (Nat Commun) PubMed Central 6e). Fluorescence was collected by the same objective and separated from the TIR beam by a dichroic mirror Di01-R405/488/561/635 (Semrock, Rochester, NY, USA). Predictions for the aptamer sequences were carried out by means of the software catRAPID omics, version 2.0, available at http://s.tartaglialab.com/page/catrapid_omics2_group (the RNA Fitness of Apt-1 is at https://tinyurl.com/y3578hr3 and the Protein Fitness of Apt-1 is at https://tinyurl.com/evmybn73). G.G.T. Sci. You are using a browser version with limited support for CSS. Purified RRM1-2 samples stored at 80C were rapidly defrosted and diluted to 20M in a high salt buffer (10mM potassium phosphate buffer pH 7.2, 150mM KCl). 30, 277288 (2011). Docking was constrained to the region of RRM1-2 in direct contact with RNA using the information available from the NMR model structure22. Fewer contacts were found for nApt-1 (number of contacts: 30, contacts distance: 8.70, fraction of H-bonds at 2.9 or below: 42%; Fig. volume13, Articlenumber:3306 (2022) ****p=1.97691011; analyzed by two-sided t-test, confidence interval 95%. Horrocks, M. H. et al. Agostini, F., Cirillo, D., Bolognesi, B. 6d was 0.89 (these values indicate a co-localization of the fluorescence associated with TDP-43 and Apt-1 of 85% and 89%, respectively). We thank the RNA Initiative at IIT, all members of the M.H.H., A.P., and G.G.T. We note that the Protein Fitness scores calculated on full-length TDP-43 have a Pearson correlation of 0.88 with Protein Fitness scores calculated for the two RRM domains (amino acids 102269, hereafter named RRM1-2) and a correlation of 0.81 with the Protein Fitness scores calculated on the RRM1 alone (negligible correlation with RRM2; Fig. In cells, Apt-1 interacts with both diffuse and condensed forms of TDP-43, indicating that Apt-1 can be exploited to follow TDP-43 phase transition. a Schematic representation of SR imaging. 45, 1288812903 (2017). PubMed Supplemental data for this article is available online at https://doi.org/10.1080/15257770.2021.1951754 . Since TDP-43 preferentially binds GU-rich RNA through its RRM domains16, we counted the number of GU repetitions in the top and bottom 100 ranked regions based on the iCLIP scores (corresponding to the strongest and poorest 1 signal sequences). We validated our binding propensity predictions demonstrating, by biolayer interferometry, that the aptamers generated by catRAPID bind to the RRM domains of TDP-43 with affinities in the nanomolar to micromolar range. Characterizing the RNA targets and position-dependent splicing regulation by TDP-43. Mol. 21, 8420 (2020). The fluorophores were excited at either 405nm (ThT), or 561nm (Atto590). Provided by the Springer Nature SharedIt content-sharing initiative. Nat. Nat. The final protein concentration was assessed by absorbance at 280nm and adjusted to 15M. 3bd). More in general, the method or its sub-parts (i.e., aptamer design and aptamer-based SR spectroscopy) may also have an important impact on drug development as well as on basic research. a Wide view of Hek293T cells co-transfect with the plasmid for the overexpression of full-length TDP-43 fused to eGFP and the aptamer nApt-1 conjugated to the fluorophore Atto590. Thus, the selected sequences had RNA Fitness that could not be further increased by mutating their nucleic acid sequences, and a specificity for TDP-43 ranging from low (Apt-6, value of 0.75; Fig. Nucleic Acids 18, 99109 (2019). 6d and S15b, c). To assess catRAPID performances (Fig. Single-molecule imaging of individual amyloid protein aggregates in human biofluids. Structural and functional analysis of the simultaneous binding of two duplex/quadruplex aptamers to human -thrombin. Vendramin, R. et al. Zacco, E., Martin, S. R., Thorogate, R. & Pastore, A. We have previously used AD-PAINT to visualize aggregates of A42 and -synuclein20, exploiting an aptamer that did not bind to a specific protein, but recognized instead the structural motif of the extended beta-sheet found in amyloid aggregates. PubMed Neither protein showed binding to Apt-1 or nApt-1 within the tested conditions, emphasizing the specificity of Apt-1 towards TDP-43. The microscope was automated using the open-source microscopy platform Micromanager (NIH, Bethesda). Med. S1; p-value 4102, Wilcoxon signed-rank test). J. Biol. In this study, we review theoretical and joint theoretical-experimental studies dedicated to aptamer design and. S13). The immobilized RRM1-2 aggregate is transiently bound by an Atto590-tagged Apt1 molecule, the position of which is determined with nanometer precision. This situation recapitulates the pathological TDP-43 behavior38. : Funding acquisition and supervision. The length of the window was set according to the number of contacts that an RNA oligonucleotide establishes with the RRM domains of TDP-43 in an NMR structure (PDB 4bs2; Fig. International journal of biological macromolecules. The reported binding curves are examples of the output of one experiment. ADS Our aptamers will provide a valuable tool for various detection methods and accelerate our understanding of TDP-43 aggregation in ALS, FTLD, and other neurodegenerative diseases. S16gi. They have the potential to bind to a wider range of targets, from ions and small molecules to proteins and large complexes. Source data are provided with this paper. Apt-1 has the highest affinity, with a Kd of 100nM (Figs. Did you know that with a free Taylor & Francis Online account you can gain access to the following benefits? Since TDP-43 is a DNA/RNA-binding protein with high tendency to form clinically relevant inclusions, we reasoned that RNA molecules could be exploited to monitor its condensation. S6; Online Methods). Chem. Similarly, Apt-1 was associated with strongly positive covariance of binding site motions (0.56) similar to those of the NMR oligonucleotide (0.76), indicating stable interactions. These results are strengthened by the Manders overlap values: e.g., 0.25, 0.21, and 0.19 for the three cells reported in Fig. Source data are provided as a Source data file64. Cells with pathological TDP-43 showed either homogenous cytosolic distribution of the protein (Fig. People also read lists articles that other readers of this article have read. The positions of the transiently immobilized Apt-1 within each frame were determined using the PeakFit plugin (an imageJ/Fiji plugin of the GDSC Single Molecule Light Microscopy package (http://www.sussex.ac.uk/gdsc/intranet/microscopy/imagej/gdsc_plugins) for imageJ using a signal strength threshold of 30 and a precision threshold of 60nm. Computational predictive approaches for interaction and structure of aptamers. Following a further affinity purification step with a nickel-coated resin, a HiTrap Heparin column was used to remove nucleic acids. Sanchez de Groot, N. et al. 6b), suggesting an interaction between soluble TDP-43 and the aptamer. In silico design and validation of high-affinity RNA aptamers targeting epithelial cellular adhesion molecule dimers. 4a, b). and A.P.] For other proteins, it might be important to design aptamers against regions that are available upon aggregation. CAS Neuron 102, 321338.e8 (2019). We observed that Apt-1 has no detectable effects on the aggregation kinetics (Fig. The exact concentration was determined by absorbance (421nm=36,000M1cm1). A set of conformers were considered as stable if the RMSD did not vary significantly in the trajectory. Magnus, M., Boniecki, M. J., Dawson, W. & Bujnicki, J. M. SimRNAweb: a web server for RNA 3D structure modeling with optional restraints. In Silico Screening of Aptamers Configuration against Hepatitis B Surface Antigen, International Journal of Molecular Sciences, Aptamers are nucleic acid analogues of antibodies with high affinity to different targets, such as cells, viruses, proteins, inorganic materials, and coenzymes. F.P.P. Sander, J., Ester, M., Kriegel, H.-P. & Xu, X. Density-based clustering in spatial databases: the algorithm GDBSCAN and its applications. 6bd and S15af) and fluorescence profiling (Figs. c Intensity profile of the oligomer imaged in (b). c Structural representation of residues with stable contacts in complex with Apt-1 (RRM1-2 representative configuration at equilibrium shown; colors correspond to panels (a) and (b). The color of the boxes follows the same as in panel (a). We have previously developed a method that can perform large-scale predictions of proteinRNA interactions. In silico designed RNA aptamer against epithelial cell adhesion molecule for cancer cell imaging. Espargar, A., Sabate, R. & Ventura, S. Thioflavin-S staining coupled to flow cytometry. 2a andS2). TDP-43 wild-type gene was cloned downstream to the eGFP gene in a pEGFP C1 mammalian transfection vector; Apt-1 was purchased with the fluorophore Atto590 at its 3 end and with the cytosines on positions 1 and 9 chemically modified with 2-Fluoro modification, to increase in-cell stability against nuclease degradation (Supplementary TableS4). We generated a positive set composed of 100 sequences with the highest iCLIP scores. Despite their undiscussed value, one of the main drawbacks of aptamers is their generation. To prioritize our candidate RNA aptamers, we calculated the RNA and Protein Fitness scores. For the microscopy studies, cells were plated on 24-well plates containing coverslips pre-treated with poly-L-lysine. The number of aggregates detected with Apt-1 was significantly higher than with nApt-1 (Fig. The eluate was dialyzed and cleaved from the His-tag by Tobacco Etch Virus protease cleavage (1:20 enzyme:protein molar ratio). PubMed Central Acta Neuropathol. Our MD simulations indicate that RRM1-2 interactions with Apt-1 (number of contacts: 38, contacts distance: 6.29, fraction of H-bonds at 2.9 or below: 50%) are comparable to those identified within the NMR structure used as reference (number of contacts: 36, contacts distance: 4.69, fraction of H-bonds at 2.9 or below: 47%). Improved in vitro aptamers screening of hepatitis B surface antigen (HBsAg) through optimization of the length sequences obtained from SELEX by minimizing the aptamer length for its easy manipulation. Rahimi, F., Murakami, K., Summers, J. L., Chen, C.-H. B. Article ACS Chem. To quantify this phenomenon, we studied the fluorescence profiles as above described and confirmed a lack of co-distribution of the two fluorophores relative to TDP-43 and nApt-1 (Fig. ISSN 2041-1723 (online). Other examples of Manders overlap calculations are reported in the SI (Fig. This agreement is, in our opinion, not casual but reflects the correctness of the initial assumptions on the forces that regulate RNAprotein interactions. Following the characterization of RRM1-2 in vitro aggregates, we proceeded to image TDP-43 condensates in the cell. Biol. K.J. & Trojanowski, J. Q. TDP-43 proteinopathy: the neuropathology underlying major forms of sporadic and familial frontotemporal lobar degeneration and motor neuron disease. Empirical approaches allow the design of in vitro aptamers that bind particularly to a target molecule with high affinity and selectivity. Source data are provided as a Source data file64. Biosyst. Experiments were repeated three times. This process is repeated to generate a SR image of each aggregate. We found a significant increase in the number of GU repeats (Fig. Successful selection heavily depends on the quality and coverage of the nucleic acid library employed2,6. E.Z. PubMed The clustered localizations were plotted as 2D Gaussian distributions, with a width equal to the precision that they were localized to. wishes to thank UCB Biopharma and Dr Jim Love for providing funding for the instrument used to generate the super-resolution data in this manuscript. Mol. This research was funded by Secretara de Investigacin y Posgrado (IP N) grant numbers 20211520 and 20211631. 1e). TDP-43 is heavily implicated in amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimers disease (AD), relentlessly progressive neurodegenerative diseases with no cure17. The experimental Kd values correlate with the predicted Protein Fitness scores computed by catRAPID (Fig. In silico approach for Post-SELEX DNA aptamers: A mini-review. b Example time montage of an oligomer being imaged using PAINT. This structure offers an ideal reference set, since it defines the interactions between the same domains of TDP-43 (RRM1-2) and a short RNA sequence natural binder of TDP-43, according to CLIP data22. e Summary of Protein Fitness scores for TDP-43 full-length, RRM1, RRM2, RRM1-2 sequences (the colors match the Protein Fitness reported in panel d). The algorithm, named catRAPID, predicts interactions between protein and RNA pairs based on the physico-chemical properties encoded in their sequences7,8. Get the most important science stories of the day, free in your inbox. We use cookies to improve your website experience. J. Microsc 169, 375382 (1993). Our starting point for the catRAPID predictions was a list of more than 3 million RNA fragments identified by iCLIP to bind TDP-4316. Biol. Vandelli, A. et al. Taken together, these data suggest that our in silico method can, in principle, provide protein-specific predictions of RNA-binding and rank the results in terms of their expected binding propensity. 141, 577585 (2014). Rep. 5, 16696 (2015). The authors declare the following competing interests: E.Z. Individual localizations appear as bursts in intensity that are separated in space and time. Adv. We felt rewarded to see that catRAPID and MD, that are based on different rationales and methodologies, converged in the analysis of Apt-1 and nApt-1. After 24h, cells were fixed and visualized by confocal microscopy (Fig. Article & Liu, S. The dataset for proteinRNA binding affinity. The emission beam was passed through a 2.5x beam expander and focussed onto an EMCCD camera for image collection (Delta Evolve 512, Photometrics, Tucson, AZ, USA) operating in frame transfer mode (EMGain=11.5 e/ADU and 250 ADU/photon). S5). Kuo, P.-H., Doudeva, L. G., Wang, Y.-T., Shen, C.-K. J. The number of contacts, distance, and numbers of H-bonds provide an estimate of the tightness of contacts29. PubMed Structural analysis of SARS-CoV-2 genome and predictions of the human interactome. Chem. Also for this type of cells, both imaging and fluorescence profiles showed almost perfect overlap between the green fluorescence of TDP-43 and the red fluorescence of Apt-1, proving that Apt-1 can recognize and bind also mislocalized and aggregated TDP-43 (Figs. The algorithm discriminates interacting and non-interacting regions with performances that increase proportionally to the iCLIP score, indicating that strong-signal regions could be accurately predicted (Fig. Aptamers, however, have a number of advantages over their protein counterparts3: once an aptamer sequence is identified, they are easier and less expensive to prepare and chemically modify. Article Thiviyanathan, V. & Gorenstein, D. G. Aptamers and the next generation of diagnostic reagents. The presence of RRM2 in tandem with RRM1 cooperatively increases the affinity, thanks to additional interactions established within RRM224. Aptamers are artificial oligonucleotides binding to specific molecular targets. Biotechnol. Mol. Buratti, E. & Baralle, F. E. Characterization and functional implications of the RNA binding properties of nuclear factor TDP-43, a novel splicing regulator of CFTR exon 9. 276, 3633736343 (2001). 31, 12601274 (2013). Fang, Y.-S. et al. Lancet Neurol. Probing TDP-43 condensation using an in silico designed aptamer, \(\pi \left(s,{{{{{{\rm{TDP}}}}}}}43\right)\), $${RNA}\,{Fitness}={{{{{{\mathscr{l}}}}}}}^{-1}\mathop{\sum }\limits_{i=1}^{{{{{{\mathscr{l}}}}}}}\theta \left[\pi \left(s,{{{{{{\rm{TDP}}}}}}}43\right)-\pi \left(i,{{{{{{\rm{TDP}}}}}}}43\right)\right]$$, $${Protein}\,{Fitness}={{{{{{\mathscr{l}}}}}}}^{-1}\mathop{\sum }\limits_{i=1}^{{{{{{\mathscr{l}}}}}}}\theta \left[\pi \left(s,{{{{{{\rm{TDP}}}}}}}43\right)-\pi \left(s,i\right)\right]$$, https://doi.org/10.1038/s41467-022-30944-x. d Histogram of precisions of the oligomer imaged in (b). Isolated RRM1-2 is purifiable as a monomer under near-to-physiological conditions and in suitable quantities for biophysical studies24. 81, 103113 (2021). : Conceptualization, catRAPID calculations, data analysis, funding acquisition, supervision, and writing. b After an initial decrease in the number of aggregates detected using Apt1 from 0 to 4h, there is a subsequent increase in their number until 48h, after which there is a slight decrease as the aggregates get larger in size (f and g). Marchese, D. et al. Further information on research design is available in theNature Research Reporting Summary linked to this article. To demonstrate the efficiency and accuracy of our method compared to ThT staining, we employed the best of our aptamers to visualize RRM1-2 aggregates with SR microscopy as they formed over 72h. Not only were we able to image individual aggregates, but we could also image them at the nanometer scale, enabling their sizes to be accurately measured. Fast flow microfluidics and single-molecule fluorescence for the rapid characterization of -synuclein oligomers. Appl. In this study we exploit the transient binding of an in silico designed aptamer, Apt-1, to TDP-43 to mimic the transient binding of the docking and imaging strands, thus allowing the use of Apt-1 labeled with an organic fluorophore for SR imaging and eliminating the two-component system of AD-PAINT. Google Scholar. We modified Apt-1 for SR microscopy, which can be employed to achieve a resolution of 5nm (Fig. 754490. Phys. CAS a Binding response curve for the interaction between RRM1-2 and Apt-1. 7). The experimental values are reported in Table1. Tsukakoshi, K., Abe, K., Sode, K. & Ikebukuro, K. Selection of DNA aptamers that recognize -synuclein oligomers using a competitive screening method. We previously demonstrated that a molar excess of the NMR oligonucleotide added to soluble RRM1-2 at the beginning of a kinetics of aggregation can reduce its propensity to undergo condensation31. The selected candidates have positive interaction propensity, RNA Fitness score of 1 and Protein Fitness>0.75. We further studied the cellular distribution of nApt-1 in cells overexpressing TDP-43, to validate the specificity of the binding in living cells and confirm the importance of tailored-designed targeting (Fig. In this article, we propose bioinformatics strategies for modeling and analysis of the interaction with molecular targets for two RNA aptamers: ATP binding RNA aptamer and iSpinach aptamer. J. Mol. Constructs were subsequently spun at 100,000g for 1h to remove any degraded or aggregated protein. Searching the Sequence Space for Potent Aptamers Using SELEX in Silico. TDP-43 regulates its mRNA levels through a negative feedback loop. Nucleic Acids Res. By closing this message, you are consenting to our use of cookies. ADS 15, 11821194 (2016). The aptamers were named after their Protein Fitness score, from 1 (0.99 score) to 6 (0.75 score). The SimRNA software was first used to generate an ensemble of initial conformers27, short MD trajectories were then run to generate structures for docking through HDOCK28 and full MD trajectories in the microsecond scale were produced (Online Methods). Google Scholar. Biosci. 6eg and S15gi) describing pixel by pixel the fluorescence intensity of DAPI, eGFP, and Atto590. 5 Howick Place | London | SW1P 1WG. Bioinforma. Compared to the other molecules, aptamers have the great advantage of a smaller molecular weight and, by means of the catRAPID design tool, easier production. Crystal structure and pair potentials: a molecular-dynamics study. S13; p-value <0.0001, two-tailed unpaired t-test) with 5 times as many aggregates. Cell Biol. PubMed PLoS ONE 4, e7694 (2009). Kwong, L. K., Neumann, M., Sampathu, D. M., Lee, V. M.-Y. Here, we demonstrate that we can rationally design aptamers able to bind a specific protein using an in silico approach. Cell Rep. 11, 17601771 (2015). Buglak, A. Struct. Nucleic Acids Res. Recommended articles lists articles that we recommend and is powered by our AI driven recommendation engine. catRAPID performances increase with the strength of the iCLIP score, indicating that the algorithm can accurately identify strong-signal interactions. To quantify the co-localization of the aptamer and its target protein, we measured the Manders overlap, which determines the co-occurrence of the two fluorescence signals while taking into account pixel intensity in a Z-stack39. Nat. ThT (Sigma) was dissolved in absolute ethanol (99%) and then diluted in PBS buffer and filtered (0.02m filtered, Anotop25, Whatman). Other most commonly used approaches for the imaging and morphology studies of protein aggregates are transmission electron microscopy (TEM)50 and atomic force microscopy (AFM)51. 14, 452458 (2011). (and Kings College London) but they can be granted upon reasonable request under non-disclosure agreement. Currently, our computational approach does not include a pipeline to predict the effect of specific chemical modifications to enhance RNA stability or avidity towards its target. As previously shown in our works, the interaction propensity calculated with catRAPID correlates with the experimental binding affinities9,10 and was successfully exploited to identify the binding partners of non-coding transcripts such as Xist8, HOTAIR11,12, SAMMSON13, in addition to interactomes of RNA genomes14. Quaternary structure defines a large class of amyloid- oligomers neutralized by sequestration. d The Protein Fitness score measures the strength of interaction propensity of each RNA sequence for TDP-43 in comparison with a pool of protein sequences with the same length and amino acid composition. 1e) and with previous literature23,24. 6, 563573 (2012). Thus, our candidate aptamers were predicted to specifically interact with RRM1-2 and RRM1. acknowledges funding from UK DRI (grant REI 3556) and AlzheimerUK (grant ARUK-PG2019B-020). Previous work indicates that TDP-43 interactions with RNA molecules span the micromolarnanomolar affinity range and tight binders contain GU repetitions16,31. In our analysis, we reported a descriptor indicating the probability of finding a H-bond within a distance cutoff of 2.9 to measure the most probable distance of the H-bonds for the aptamer-RRM1-2 complex. Full-length TDP-43 typically forms oligomers of 101000nm32, but, due to the diffraction-limit of light, their visualization is restricted to 250nm (Fig. Bellucci, M., Agostini, F., Masin, M. & Tartaglia, G. G. Predicting protein associations with long noncoding RNAs. Neither Apt-1 nor nApt-1 was able to exhibit substantial binding to either protein, meaning that a low number of aggregates could be detected using this approach (Fig. Nature 344, 467468 (1990). By considering the RNA and Protein Fitness scores, we prioritized a set of RNA sequences: six candidate aptamers were selected with an RNA Fitness score of 1 and a Protein Fitness score ranging from 0.75 to 0.99 (Fig. Centre for Human Technologies (CHT), Istituto Italiano di Tecnologia (IIT), Via Enrico Melen, 83, 16152, Genova, Italy, Elsa Zacco,Alexandros Armaos,Stefano Gustincich&Gian Gaetano Tartaglia, EaStCHEM School of Chemistry, University of Edinburgh, Edinburgh, EH9 3FJ, UK, Owen Kantelberg,Kiani Jeacock,David J. Clarke&Mathew H. Horrocks, Department of Physics, Sapienza University, Piazzale Aldo Moro 5, 00185, Rome, Italy, Edoardo Milanetti,Francesco Paolo Panei&Giancarlo Ruocco, Center for Life Nanoscience, Istituto Italiano di Tecnologia, Viale Regina Elena 291, 00161, Rome, Italy, UK Dementia Research Institute at University of Edinburgh, University of Edinburgh, Edinburgh bioQuarter, Chancellors Building, 49 Little F, Edinburgh, UK, Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, UK, Euan MacDonald Centre for MND Research, University of Edinburgh, Edinburgh, UK, UK Dementia Research Institute at the Maurice Wohl Institute of Kings College London, London, SE5 9RT, UK, Centre for Genomic Regulation (CRG), Dr. Aiguader 88, 08003, Barcelona, Spain, Catalan Institution for Research and Advanced Studies, ICREA, Passeig Llus Companys 23, 08010, Barcelona, Spain, Department of Biology Charles Darwin, Sapienza University of Rome, P.le A. Moro 5, Rome, 00185, Italy, You can also search for this author in Both techniques provide qualitative and quantitative information at the nanometer level, but the former is limited in resolution and the latter requires long and complex sample preparation. 1e). received funding from the Newton fellowship scheme and the MINDED fellowship of the European Unions Horizon 2020 research and innovation program under the Marie Skodowska-Curie grant agreement No. We validated the aptamer binding affinities by measuring the dissociation constants (Kd) through bio-layer interferometry (BLI). The super-resolution and SAVE image data generated in this study have been deposited in the Zenodo database under accession code 6533779. CAS S4). S4). Zacco, E. et al. 214, 34813495 (2017). The images or other third party material in this article are included in the articles Creative Commons license, unless indicated otherwise in a credit line to the material. Neurodegenerative diseases: quantitative predictions of protein-RNA interactions. Proteins 11, 205217 (1991). bd Examples of isolated cells co-transfected as in (a), in which the lack of correlation between green and red fluorescence distribution is evident. Article Olzscha, H. et al. The catRAPID algorithm is exploited to generate aptamers targeting TAR DNA-binding protein 43, whose aggregation is associated with Amyotrophic Lateral Sclerosis, and it is demonstrated that they bind TDP-43 and used the tightest interactor, Apt-1, as a probe to visualize T DP-43 condensates with super-resolution microscopy. S.C.: Funding acquisition, supervision, and writing. The SAVE images were first thresholded using a value of intensity mean+2 S.D., and then analyzed using the measure module (skimage v0.18.1). Thus, exploiting an aptamer that interacts with TDP-43 in the soluble state is effective to monitor the evolution of assemblies over time. All details relative to the sequences, type, and positions of the aptamers employed in this work can be found in the Supplementary Information (TableS4). c The RNA Fitness score was employed to measure the strength of catRAPID interaction propensities upon mutation of the RNA fragment sequence. 4d)33. 22, 18081811 (2013). Full-length TDP-43 forms toxic amyloid oligomers that are present in frontotemporal lobar dementia-TDP patients. An effort to present the potential of computational techniques in aptamer selection, a simple sequence-based method was used to design a 69-nucleotide long aptamer with a relatively stable structure and investigate its binding properties to the tyrosine kinase domain of the NT-3 growth factor receptor, for the first time, by employing computational modeling and docking tools. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. For each list of fragments, we computed the RNA Fitness and Protein Fitness scores using catRAPID (Online Methods). Unlike AD-PAINT20, which requires the aptamer to be modified with a short DNA sequence for DNA-PAINT, the affinity of Apt-1 to RRM1-2 meant that it was possible to localize and build up a SR image as the labeled aptamer transiently binds to the aggregates (Fig. nApt-1 was purchased with the fluorophore Atto590 at its 5 end and with the guanines on positions 1 and 9 chemically modified with 2-Fluoro modification, to increase in-cell stability against nuclease degradation (Supplementary TableS4). a Wide view of Hek293T cells co-transfected with the plasmid for the overexpression of full-length TDP-43 fused to eGFP and the aptamer Apt-1 conjugated to the fluorophore Atto590. The striking co-occurrence of Apt-1 and TDP-43, resulting into a yellow color derived from the overlap between the red fluorescence of Apt-1 and the green fluorescence of TDP-43, was even more evident in the 3D images derived from the Z-stacks of the analyzed cells (Fig. RNA as a key factor in driving or preventing self-assembly of the TAR DNA-binding protein 43. We have previously developed an SR method that makes use of aptamers to image targets with nanometer resolution20.
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