For other proteins, it might be important to design aptamers against regions that are available upon aggregation. Nanoscopic characterisation of individual endogenous protein aggregates in human neuronal cells. http://www.sussex.ac.uk/gdsc/intranet/microscopy/imagej/gdsc_plugins, http://s.tartaglialab.com/page/catrapid_omics2_group, https://doi.org/10.1038/s41594-018-0143-4, http://creativecommons.org/licenses/by/4.0/. Correspondence to Tuerk, C. & Gold, L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. 431, 16711688 (2019). HDOCK: a web server for protein-protein and protein-DNA/RNA docking based on a hybrid strategy. was funded via the BBSRCEastBIO doctoral training program (BB/M010996/1). Rea, J. et al. Having shown that Apt-1 is able to bind to RRM1-2, we next explored the possibility of using this aptamer as imaging probe to monitor the aggregation of TDP-43. To learn about our use of cookies and how you can manage your cookie settings, please see our Cookie Policy. 1a; Online Methods), we generated a list of fragment sequences using a window of 10 nucleotides moved from the 5 to the 3 of each sequence (TableS1). This is interesting because it is known that RRM1 is necessary and sufficient for the interaction, whereas RRM2 is unable to bind in isolation23,25. Science 341, 655658 (2013). The color of the boxes follows the same as in panel (a). Cirillo, D. et al. The de novo generation of aptamers and their use for microscopy opens a new page to study protein condensation. Calculations for sequences <50 nucleic acids are restricted in the webserver due to the filing of a patent (see also Data availability) but they can be provided upon reasonable request under non-disclosure agreement. Mann, J. R. et al. Article As a control, we investigated the interaction of Apt-1 and of nApt-1 with two more amyloidogenic proteins in their soluble forms: A42 and -synuclein (Fig. Recently, numerous techniques, grouped under the umbrella term of super-resolution (SR) microscopy19, have been developed to surpass this limit, enabling imaging at a resolution as high as 5nm. Magnus, M., Boniecki, M. J., Dawson, W. & Bujnicki, J. M. SimRNAweb: a web server for RNA 3D structure modeling with optional restraints. Basel Switz. PLoS ONE 4, e7694 (2009). Bell, D. R. et al. 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. ChemBioChem 19, 20332038 (2018). 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. Appl. Cited by lists all citing articles based on Crossref citations.Articles with the Crossref icon will open in a new tab. These results validate the computational design of RNA aptamers and indicate Apt-1 as the sequence with the highest affinity for TDP-43. Neurosci. S6; Online Methods). G.G.T. Starting from 1s, we prolonged the simulation of 1s to confirm that the RMSD had small fluctuations (<1) in at least the 60% of the trajectory. Zacco, E., Martin, S. R., Thorogate, R. & Pastore, A. S15df and jl. Hydrogen bonds (H-bonds) were calculated using Gromacs default function62,63. ADS At 72h, only 6% of the aggregates detected using Apt1 are ThT-active. Briefly, after cell lysis and centrifugation, the protein fractions were resuspended and passed through a nickel-coated resin. S3). Z-stacks for selected cells were composed acquiring scans every 0.5m for 6m above and below the median plane. bd Examples of isolated cells co-transfected as in (a), in which the lack of correlation between green and red fluorescence distribution is evident. Google Scholar. Simulations were carried out setting the system temperature to 300K. The equilibrium condition was evaluated based on the root mean square deviation (RMSD). Following TDP-43 distribution, cells were found in a mixed population composed of elements with nuclear distribution of TDP-43, and cells with mislocalized and/or condensed cytosolic TDP-43 (Fig. Design and functional validation of a mutant variant of the LncRNA HOTAIR to counteract snail function in epithelial-to-mesenchymal transition. ISSN 2041-1723 (online). 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. Clin. Aging Cell 20, e13326 (2021). A significant improvement of the affinity for almost all aptamers was observed with RRM1-2. This situation recapitulates the pathological TDP-43 behavior38. Chem. E.Z. Other examples of the poor or absent co-localization between TDP-43 and nApt-1 are given in Figs. Article 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. Disco. 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). Nuclear pore scaffold structure analyzed by super-resolution microscopy and particle averaging. : Conceptualization, data analysis, funding acquisition, supervision, and writing. Struct. Searching the Sequence Space for Potent Aptamers Using SELEX in Silico. Non-His-tagged proteins were washed through and the constructs were eluted with high imidazole concentration (10mM potassium phosphate buffer pH 7.2, 150mM KCl, 250mM imidazole). 6a and S14; green and red fluorescence appeared to be present in the same cells, hinting at an interaction between the protein and the RNA aptamer). 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. Cells with nuclear TDP-43 also displayed nuclear distribution of Apt-1 (Fig. The repeated binding and unbinding of multiple probes allow SR to be achieved. Cordeiro, Y., Macedo, B., Silva, J. L. & Gomes, M. P. B. Pathological implications of nucleic acid interactions with proteins associated with neurodegenerative diseases. M.H.H. 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. J. Microsc 169, 375382 (1993). The top iCLIP score regions (30 sequences) were divided into 10 nucleotide fragments (260 sequences). 3b; Online Methods) shows that nApt-1 binds less well because the contacts are formed in highly mobile regions (amino acids 144 and 267, corresponding to loops), whereas Apt-1 interacts with elements with lower flexibility (amino acids 135, 145, 255, and 263, Fig. We first hypothesized that catRAPID, a software that has been already proven to successfully identify RNAprotein interactions7,8, could be used to design RNA aptamers against TDP-43. 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. 45, 11961199 (1980). Mislocalization of nuclear TDP-43 to the cytoplasm and its subsequent aggregation into toxic inclusions is a common pathological feature in over 97% of all ALS cases18. : MD calculations. Nucleic Acids Res. Cell Death Dis. However, this procedure requires considerable time and cost. Google Scholar. Google Scholar. Atto590-tagged Apt-1 (sequence provided in Supplementary TableS4) was diluted in PBS buffer and used at a final imaging concentration of 1nM. 45, W365W373 (2017). acknowledges funding from UK DRI (grant REI 3556) and AlzheimerUK (grant ARUK-PG2019B-020). Aliquots were taken at given time points (0, 4, 8, 12, 24, 48, 72h) and flash frozen for later analysis. 3099067 Google Scholar. Ranking from moderate- (100 transcriptomic regions with highest iCLIP score compared with 100 transcriptomic regions with the lowest iCLIP score) to strong-signal experimental data (20 transcriptomic regions with the highest iCLIP score compared with 20 transcriptomic regions with the lowest iCLIP score), we found that the AUC increases progressively reaching a maximal value of 0.89, which indicates that catRAPID accurately identifies strong-signal interactions. 31, 12601274 (2013). 11, 372 (2018). 6, 563573 (2012). CAS catRAPID performances increase with the strength of the iCLIP score, indicating that the algorithm can accurately identify strong-signal interactions. International journal of biological macromolecules. Milordini, G. et al. was supported by a Scottish PhD Research & Innovation Network Traineeships in MND/MS. S4). A short single-stranded docking DNA oligonucleotide is attached to a target-specific probe capable of binding to the molecule-of-interest. Rahimi, F., Murakami, K., Summers, J. L., Chen, C.-H. B. The RNA Fitness score was defined as the value obtained by randomly introducing mutations in the RNA sequences to alter the interaction propensity for TDP-43 (Fig. Following a further affinity purification step with a nickel-coated resin, a HiTrap Heparin column was used to remove nucleic acids. We use cookies to improve your website experience. 1b; Online Methods)22. EMBO J. Nature 346, 818822 (1990). a Representative Apt-1 SR images (left, red hot) and diffraction-limited SAVE images (right, gray) of RRM1-2 species over a 72h aggregation assay, scale bar: 500nm. Lett. The aptamers were simulated in water to explore the conformational space and to select extended structures that would preferably interact with TDP-43 based on previous knowledge22. RNA structure drives interaction with proteins. Confocal microscopy analysis of cells expressing TDP-43 and transfected with Apt-1 indicate that this aptamer interacts with TDP-43 both in the soluble and condensed forms. Interestingly, only 6% of the aggregates detected using Apt-1 were ThT-active, also at later time points, demonstrating the aptamers ability to identify less mature oligomers that may be important in the disease pathology of TDP-43 and overcoming the limitations of current methods, such as the identification of oligomers of different size and structure. 2c; Fig. The coupling system of the modified Berendsen thermostat was applied with pressure set at 1bar, following Parrinello-Rahman method61. 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. A large-scale binding and functional map of human RNA-binding proteins. & Trojanowski, J. Q. TDP-43 proteinopathy: the neuropathology underlying major forms of sporadic and familial frontotemporal lobar degeneration and motor neuron disease. View 2 excerpts, cites background and methods, Journal of molecular graphics & modelling. Sketch of the computational pipeline. Empirical approaches allow the design of in vitro aptamers that bind particularly to a target molecule with high affinity and selectivity. Thiviyanathan, V. & Gorenstein, D. G. Aptamers and the next generation of diagnostic reagents. S1; p-value 4102, Wilcoxon signed-rank test). We used SimRNA webserver27 to generate a set of initial structures for Apt-1 and nApt-1. https://doi.org/10.1038/s41594-018-0143-4 (2018). 2a andS2). Each sub-image is separated by 1s, moving through time from left to right then top to bottom; scale bar: 400nm. Glass slides with fixed cells were visualized with a Nikons A1R MP multiphoton confocal microscope, employing the 60 objective and 3 channel non-descanned detectors. Steps towards this direction usually exploit the use of fluorescent proteins, such as GFP, fused to the protein target to identify localization and condensation within the cell. 7). Proteom. Nat. Apt-1 and nApt-1 were purchased from Sigma-Merck. This approach, however, has several limitations: it needs careful planning, specific expertise, and is costly and time consuming. Kd values were estimated by fitting the response intensity (shift in the wavelength upon binding) as a function of the protein concentration, at the steady state. In summary, our ability to generate aptamers de novo and use them for microscopy opens a new page in the field of high-resolution molecular imaging to study the evolution of aggregates in tissues. c Experimental Kd and Protein Fitness scores are tightly anti-correlated (Pearson correlation of 0.93). These authors contributed equally: Elsa Zacco, Owen Kantelberg. Nucleic Acids Res. Each system was simulated by MD, starting at room temperature and progressively increasing the temperature until the percentage of extended conformers corresponded to at least 10% of the total possible structures. The super-resolution and SAVE image data generated in this study have been deposited in the Zenodo database under accession code 6533779. Liu, P. et al. CAS ACS Chem. Mol. 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. In addition, we verified, in silico and in vitro, the structural similarity between Apt-1 and the short RNA reported in the NMR structure and confirmed Apt-1 to be linear (Fig. 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. 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. We found a significant increase in the number of GU repeats (Fig. Biosci. Novel Design of RNA Aptamers as Cancer Inhibitors and Diagnosis Targeting the Tyrosine Kinase Domain of the NT-3 Growth Factor Receptor Using a Computational Sequence-Based Approach, In-Silico Selection of Aptamer Targeting SARS-CoV-2 Spike Protein, Artificial Intelligence in AptamerTarget Binding Prediction. Cell. The role of glycation on the aggregation properties of IAPP. 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. Structural analysis of SARS-CoV-2 genome and predictions of the human interactome. View 4 excerpts, cites methods and background, International journal of molecular sciences. 15, 45 (2020). Experiments were repeated three times. Each localization is accurate positioned with a precision of 60nm or less. The immobilized RRM1-2 aggregate is transiently bound by an Atto590-tagged Apt1 molecule, the position of which is determined with nanometer precision. In vitro production and selection of aptamers can be performed using the SELEX method. Aptamers are artificial oligonucleotides binding to specific molecular targets. The aptamers were named after their Protein Fitness score, from 1 (0.99 score) to 6 (0.75 score). 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. Nature Communications (Nat Commun) Article Molecular basis of UG-rich RNA recognition by the human splicing factor TDP-43. Zacco, E., Kantelberg, O., Milanetti, E. et al. By contrast, nApt-1 showed a negative correlation (0.53), indicating asynchronous motions and more unstable interactions (Table2). RNA as a key factor in driving or preventing self-assembly of the TAR DNA-binding protein 43. The microscope was automated using the open-source microscopy platform Micromanager (NIH, Bethesda). The negative control has a Kd of 1.52.0M (Figs. Single-molecule imaging was carried out using a custom-built TIRF microscope, restricting excitation of fluorophores within the sample to 200nm from the sample-coverslip interface. Data Min. Biol. Yan, Y., Zhang, D., Zhou, P., Li, B. This research was funded by Secretara de Investigacin y Posgrado (IP N) grant numbers 20211520 and 20211631. Fast flow microfluidics and single-molecule fluorescence for the rapid characterization of -synuclein oligomers. This proves that Apt-1 is a suitable ex-situ imaging probe for RRM1-2 aggregation without disturbing their assembly. 6). PubMed 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. 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. Med. Neither protein showed binding to Apt-1 or nApt-1 within the tested conditions, emphasizing the specificity of Apt-1 towards TDP-43. PubMed and A.P.] The authors would also like to acknowledge the help and support received during confocal images acquisition by the group of Giuseppe Vicidomini at the Molecular Microscopy and Spectroscopy Department of IIT. We note that Apt-1, designed to interact with RRM1-2, is still sensitive toward oligomers of full-length TDP-43 in the cellular context. Agostini, F., Cirillo, D., Bolognesi, B. eg Fluorescence profiles of DAPI, eGFP, and Atto590 along a diagonal line drew across the isolated correspondent cells on the left, showing corresponding distribution of the fluorescence signals of eGFP_TDP-43 and Apt-1_Atto590. Human stem cell-derived astrocytes replicate human prions in a PRNP genotype-dependent manner. 4ce). Van Nostrand, E. L. et al. : Protein purification and data analysis. Quaternary structure defines a large class of amyloid- oligomers neutralized by sequestration. Along the pathway to forming such insoluble inclusions, TDP-43 adopts a highly heterogenous population of phase-separated assemblies with differing sizes and structures, which are often smaller than the diffraction limit of light, thus limiting their characterization by standard fluorescence microscopy. 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. Krejciova, Z. et al. f Sample fields of view of clustered RRM1-2 aggregates imaged with Apt-1 and nApt-1, scale bar 1m. 41, e31 (2013). 7eg). The diffraction-limit of light restricts optical microscopy to a resolution of ~250nm. PubMed Central J. Mol. 6b), suggesting an interaction between soluble TDP-43 and the aptamer. Yang, X., Li, H., Huang, Y. Starting from RNA sequences occurring in the human transcriptome and through in silico prioritization, catRAPID identified RNA regions whose GU content increases with the binding propensity for TDP-43, thus mimicking natural TDP-43 binding partners40. Other approaches to design aptamers purely based on MD and docking46,47 cannot be straightforwardly used to perform high-throughput analyses due to the heavy amount of calculations. Biol. At the start of the aggregation reaction, pre-aggregates of RRM1-2 were detected; however, these decreased in number in the first 4h of the aggregation reaction (for example Apt-1 and SAVE images, see Fig. b Example time montage of an oligomer being imaged using PAINT. To prioritize our candidate RNA aptamers, we calculated the RNA and Protein Fitness scores. 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. Rev. We used molecular dynamics (MD) to investigate the structural mechanism of TDP-43 interaction with Apt-1 and its control nApt-1 (TableS2). As a proof-of-concept, we demonstrate that we can generate high-affinity RNA aptamers targeting the TAR DNA-binding protein 43 (TDP-43)15. 3bd). Nature Communications The corresponding 3D images can be found in Fig. In this study, we review theoretical and joint theoretical-experimental studies dedicated to aptamer design and. J. Mol. A screening tool to detect in vivo protein aggregation. After further washing in PBS, coverslips were placed faced down on glass slides using the mounting medium ProLong Diamond Antifade Mountant (Invitrogen). a TDP-43 interaction propensity computed with catRAPID is used to discriminate between interacting (high iCLIP score) and non-interacting (low iCLIP scores) transcriptomic regions. Yet, Apt-1 interacts more frequently with amino acids 104112,135139,144150, 194199, 255261, and 263264 (Fig. Nucleic Acids Res. 5, 4824 (2014). https://doi.org/10.1038/s41467-022-30944-x, DOI: https://doi.org/10.1038/s41467-022-30944-x. 2b andS2), which is in the range of values obtained for Apt-4, Apt-5, and Apt-6 (Table1). 6e). 1e; Pearson correlation of 0.93). 10, 3246 (2019). 4d)33. We exploited Apt-1 to track TDP-43 aggregation (Fig. In summary, MD confirms that Apt-1 binds tightly to RRM1-2 in the same regions that were previously reported in NMR models, thus supporting the reliability of catRAPID predictions. Single-molecule imaging of individual amyloid protein aggregates in human biofluids. Docking was constrained to the region of RRM1-2 in direct contact with RNA using the information available from the NMR model structure22. Constructs were subsequently spun at 100,000g for 1h to remove any degraded or aggregated protein. 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. Our approach thus provides a competitive high-throughput method alternative to experimental approaches such as SELEX. 4a, b). The mean number of localizations per aggregate (Fig. 5d) (Fig. More in general, our in silico design method provides a unique tool to assist quite different applications and may become a standard technique to select RNA aptamers offering, for instance, a new perspective for drug design. Importantly, RRM1-2 represents the region necessary for RNA-binding with high affinity22. 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. 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. Paul, A. et al. 21, 8420 (2020). 6eg and S15gi) describing pixel by pixel the fluorescence intensity of DAPI, eGFP, and Atto590. 7. : Conceptualization, data analysis, funding acquisition, supervision, and writing. Lukavsky, P. J. et al. 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. Provided by the Springer Nature SharedIt content-sharing initiative. 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). CAS and INFORE n. 825080 to G.G.T.] a Schematic representation of SR imaging. 87, 88188826 (2015). In silico approach for Post-SELEX DNA aptamers: A mini-review. 24h after transfection, cells were washed with phosphate-buffered saline (PBS) solution and fixed with 4% paraformaldehyde for 10min at room temperature. ThT (Sigma) was dissolved in absolute ethanol (99%) and then diluted in PBS buffer and filtered (0.02m filtered, Anotop25, Whatman). Jungmann, R. et al. K.J. As a measure of coordinated motion, we calculated the covariance among the amino acids of RRM1-2-binding site (BS), under the hypothesis that systems with higher binding affinity are characterized by strongly coordinated motions. Thus, our candidate aptamers were predicted to specifically interact with RRM1-2 and RRM1. 45, 1288812903 (2017). Protein Soc. Commun. Knowl. We demonstrated that our aptamers bind TDP-43 and used the tightest interactor, Apt-1, as a probe to visualize TDP-43 condensates with super-resolution microscopy. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. : Funding acquisition and supervision. The algorithm, named catRAPID, predicts interactions between protein and RNA pairs based on the physico-chemical properties encoded in their sequences7,8. Our starting point for the catRAPID predictions was a list of more than 3 million RNA fragments identified by iCLIP to bind TDP-4316. Proteins 11, 205217 (1991). Biol. Aptamer design was carried out through a novel approach based on the catRAPID method to predict proteinRNA interactions1,2. Supplemental data for this article is available online at https://doi.org/10.1080/15257770.2021.1951754 . Cell Biol. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Mol. Article Sci. Amyloid-like aggregates sequester numerous metastable proteins with essential. Article a Binding response curve for the interaction between RRM1-2 and Apt-1. Lancet Neurol. 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). PubMed Tosatto, L. et al. Whiten, D. R. et al. Horrocks, M. H., Palayret, M., Klenerman, D. & Lee, S. F. The changing point-spread function: single-molecule-based super-resolution imaging. & Bitan, G. RNA aptamers generated against oligomeric A40 recognize common amyloid aptatopes with low specificity but high sensitivity. Zooming in on some examples of these cells, the sub-cellular distribution of TDP-43 and Apt-1 was analyzed by imaging (Figs. 12, 11981228 (2017). 6bd and S15af) and fluorescence profiling (Figs. 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. USA 117, 84868493 (2020). To assess catRAPID performances (Fig. 84, 55425547 (2012). 1a), we considered the Area Under the Curve (AUC) of the Receiver Operating Characteristic (ROC) curve generated using iCLIP data and their catRAPID scores. For this purpose, molecular modeling of the tertiary structure of the aptamers was performed with two servers (SimRNA and RNAComposer); and AutoDock Vina and rDock programs were used to dock their respective ligands. The data shown are meanSD of 9 fields of view.
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