A novel artificial intelligence-based approach for identification of deoxynucleotide aptamers.

The selection of a DNA aptamer through the Systematic Evolution of Ligands by EXponential enrichment (SELEX) method involves multiple binding steps, in which a target and a library of randomized DNA sequences are mixed for selection of a single, nucleotide-specific molecule. Usually, 10 to 20 steps are required for SELEX to be completed. Throughout this process it is necessary to discriminate between true DNA aptamers and unspecified DNA-binding sequences. Thus, a novel machine learning-based approach was developed to support and simplify the early steps of the SELEX process, to help discriminate binding between DNA aptamers from those unspecified targets of DNA-binding sequences. An Artificial Intelligence (AI) approach to identify aptamers were implemented based on Natural Language Processing (NLP) and Machine Learning (ML). NLP method (CountVectorizer) was used to extract information from the nucleotide sequences. Four ML algorithms (Logistic Regression, Decision Tree, Gaussian Naïve Bayes, Support Vector Machines) were trained using data from the NLP method along with sequence information. The best performing model was Support Vector Machines because it had the best ability to discriminate between positive and negative classes. In our model, an Accuracy (A) of 0.995, the fraction of samples that the model correctly classified, and an Area Under the Receiving Operating Curve (AUROC) of 0.998, the degree by which a model is capable of distinguishing between classes, were observed. The developed AI approach is useful to identify potential DNA aptamers to reduce the amount of rounds in a SELEX selection. This new approach could be applied in the design of DNA libraries and result in a more efficient and faster process for DNA aptamers to be chosen during SELEX.

Interaction of metallocene dichlorides with apo-human transferrin: A spectroscopic study and cytotoxic activity against human cancer cell lines.

Metallocene dichlorides (Cp2M(IV)Cl2) are the first class of small and hydrophobic organometallic compounds classified as anticancer agents against numerous cancer cell lines and tumors. In this study, the antiproliferative activities of Cp2VCl2,Cp2NbCl2, Cp2HfCl2 and Cp2ZrCl2were assessed on two human cancer cell lines (HT-29 and MCF-7) using MTT assay. Spectroscopic studies were also conducted using these and other known metallocene dichlorides on apo-human transferrin (apo-hTf) at pH 7.4. UV-Vis and CD showed that their interaction with apo-hTf could induce conformational changes of its secondary structure during binding process. In fluorescence, a decrease in intensity of the emission peak was observed when the apo-hTf:Cp2M(IV)Cl2 complex is being formed, probably due to changes in the microenvironment of its tyrosine and tryptophan residues. Among all metallocene dichlorides studied, Cp2VCl2 has the strong ability to quench the intrinsic fluorescence of apo-hTf through a static quenching mechanism. The association constants for each protein-compound complex were also determined at different temperatures (296 K, 303 K, 310 K, and 317 K) based on fluorescence quenching results. Positive enthalpy changes (ΔH) and entropy changes (ΔS) as well as negative free energies (ΔG) suggest that hydrophobic interactions are the main intermolecular forces involved in the binding process, probably via an endothermic and spontaneous reaction mechanism. The distance, r, between donor (apo-hTf) and acceptor (Cp2M(IV)Cl2) obtained according to Forster's theory of non-radiation energy transfer suggest that the energy transfer from apo-hTf to Cp2M(IV)Cl2 occurs with high probability and distances obtained by FRET with high accuracy.

Fast Adhesion of Gold Nanoparticles (AuNPs) to a Surface Using Starch Hydrogels for Characterization of Biomolecules in Biosensor Applications.

Gold nanoparticles (AuNPs) are the most thoroughly studied nanoparticles because of their remarkable optical properties. Color changes in assays that use AuNPs can be easily observed with the naked eye, resulting in sensitive colorimetric methods, useful for detecting a variety of biological molecules. However, while AuNPs represent an excellent nano-platform for developing analytical methods for biosensing, there are still challenges that must be overcome before colloidal AuNPs formulation can be successfully translated into practical applications. One of those challenges is the ability to immobilize AuNPs in a solid support. There are many difficulties with controlling both the cluster size and the adhesion of the coatings formed. In addition, many of the techniques employed are expensive and time-consuming, or require special equipment. Thus, a simple and inexpensive method that only requires common lab equipment for immobilizing AuNPs on a surface using Starch Hydrogels has been developed. Starch hydrogels confer a 400% increase in stability to the nanoparticles when exposed to changes in the environment while also allowing for macromolecules to interact with the AuNPs surface. Several starch derivatives were tested, including, dextrin, beta-cyclodextrin and maltodextrin, being dextrin the one that conferred the highest stability. As a proof-of-concept, a SlipChip microfluidic sensor scheme was developed to measure the concentration of DNA in a sample. The detection limit of our biosensor was found to be 25 ng/mL and 75 ng/mL for instrument and naked eye detection, respectively.

Modelling and molecular docking studies of the cytoplasmic domain of Wsc-family, full-length Ras2p, and therapeutic antifungal compounds.

Saccharomyces cerevisiae, the budding yeast, must remodel initial cell shape and cell wall integrity during vegetative growth and pheromone-induced morphogenesis. The cell wall remodeling is monitored and regulated by the cell wall integrity (CWI) signaling pathway. Wsc1p, together with Wsc2p and Wsc3p, belongs to a family of highly O-glycosylated cell surface proteins that function as stress sensors of the cell wall in S. cerevisiae. These cell surface proteins have the main role of activating the CWI signaling pathway by stimulating the small G-protein Rho1p, which subsequently activates protein kinase C (Pkc1p) and a mitogen activated protein (MAP) kinase cascade that activates downstream transcription factors of stress-response genes. Wsc1p, Wsc2p, and Wsc3p possess a cytoplasmic domain where two conserved regions of the sequence have been assessed to be important for Rom2p interaction. Meanwhile, other research groups have also proposed that these transmembrane proteins could support protein-protein interactions with Ras2p. Molecular structures of the cytoplasmic domain of Wsc1p, Wsc2p and Wsc3p were generated using the standard and fully-automated ORCHESTAR procedures provided by the Sybyl-X 2.1.1 program. The tridimensional structure of full length Ras2p was also generated with Phyre2. These protein models were validated with Procheck-PDBsum and ProSA-web tools and subsequently used in docking-based modeling of protein-protein and protein-compound interfaces for extensive structural and functional characterization of their interaction. The results retrieved from STRING 10.5 suggest that the Wsc-family is involved in protein-protein interactions with each other and with Ras2p. Docking-based studies also validated the existence of protein-protein interactions mainly between Motif I (Wsc3p > Wsc1p > Wsc2p) and Ras2p, in agreement with the data provided by STRING 10.5. Additionally, it has shown that Calcofluor White preferably binds to Wsc1p (-9.5 kcal/mol), meanwhile Caspofungin binds to Wsc3p (-9.1 kcal/mol), Wsc1p (-9.1 kcal/mol) and more weakly Wsc2p (-6.9 kcal/mol). Thus, these data suggests Caspofungin as a common inhibitor for the Wsc-family. MTiOpenScreen database has provided a list of new compounds with energy scores higher than those compounds used in our docking studies, thus suggesting these new compounds have a better affinity towards the cytoplasmic domains and Ras2p. Based on these data, there are new and possibly more effective compounds that should be considered as therapeutic agents against yeast infection.

A molecular docking study of the interactions between human transferrin and seven metallocene dichlorides.

Human Transferrin (hTf) is a metal-binding protein found in blood plasma and is well known for its role in iron delivery. With only a 30% of its capacity for Fe+3 binding, this protein has the potential ability to transport other metal ions or organometallic compounds from the blood stream to all cell tissues. In this perspective, recent studies have described seven metallocene dichlorides (Cp2M(IV)Cl2, M(IV)=V, Mo, W, Nb, Ti, Zr, Hf) suitable as anticancer drugs and less secondary effects than cisplatin. However, these studies have not provided enough data to clearly explain how hTf binds and transports these organometallic compounds into the cells. Thus, a computational docking study with native apo-hTf using Sybyl-X 2.0 program was conducted to explore the binding modes of these seven Cp2M(IV)Cl2 after their optimization and minimization using Gaussian 09. Our model showed that the first three Cp2M(IV)Cl2 (M(IV)=V, Mo, W) can interact with apo-hTf on a common binding site with the amino acid residues Leu-46, Ile-49, Arg-50, Leu-66, Asp-69, Ala-70, Leu-72, Ala-73, Pro-74 and Asn-75, while the next four Cp2M(IV)Cl2 (M(IV)=Nb, Ti, Zr, Hf) showed different binding sites, unknown until now. A decreasing order in the total score (equal to -log Kd) was observed from these docking studies: W (5.4356), Mo (5.2692), Nb (5.1672), V (4.5973), Ti (3.6529), Zr (2.0054) and Hf (1.8811). High and significant correlation between the affinity of these seven ligands (metallocenes) for apo-hTf and their bond angles CpMCp (r=0.94, p<0.01) and Cl-M-Cl (r=0.95, p<0.01) were observed, thus indicating the important role that these bond angles can play in ligand-protein interactions. Fluorescence spectra of apo-hTf, measured at pH 7.4, had a decrease in the fluorescence emission spectrum with increasing concentration of Cp2M(IV)Cl2. Experimental data has a good correlation between KA (r=0.84, p=0.027) and Kd (r=0.94, p=0.0014) values and the calculated total scores obtained from our docking experiments. In conclusion, these results suggest that the seven Cp2M(IV)Cl2 used for this study can interact with apo-hTf, and their affinity was directly and inversely proportional to their bond angles CpMCp and ClMCl, respectively. Our docking studies also suggest that the binding of the first three Cp2M(IV)Cl2 (M(IV)=V, Mo, W) to hTf could abrogate the formation of the hTf-receptor complex, and as a consequence the metallocene-hTf complex might require another transport mechanism in order to get into the cell.

Electrophoretic mobility-shift and super-shift assays for studies and characterization of protein-DNA complexes.

Gene expression is in part regulated by transcription factors that bind specific sequence motifs in genomic DNA. Transcription factors cooperate with the basal machinery to upregulate or downregulate transcription. Experimental data have revealed the importance of interactions among members of distinct families of transcription factors to form complexes that regulate gene expression. Thus, a full characterization of protein-DNA complexes is essential to understanding of gene regulation in a more complex cellular environment. Electrophoretic mobility shift assay (EMSA) is a powerful technique to resolve nucleic acid-protein complexes formed with transcription factors in nuclear extracts. Herein is described how EMSA and super-shift assays were used to characterize several complexes produced from binding of transcription factors to a regulatory DNA sequence upstream from the promoter region of the human NF-IL6 gene.

Exploring transcription factor binding properties of several non-coding DNA sequence elements in the human NF-IL6 gene.

We examined several DNA segments upstream of the transcription start site of the human NF-IL6 gene to evaluate the predictions of two computational models developed to identify potential regulatory elements in the non-coding regions of genes. One model, comparative genomics, is based on the hypothesis that functional regulatory sequences can be localized in alignments of genomic DNA from several species. The other model is based on the hypothesis that protein-binding sites in genomic DNA may include sequence elements that occur frequently in proximal promoters of genes. The segments selected for DNA binding and functional evaluations included: (1) two conserved regions identified in multi-species sequence alignments; (2) a region containing several localized hits with 9-mers that ranked highly in studies of proximal promoters of human genes; and (3) two regions that were either GC-rich and/or contained tracts of G. The assays were done under nearly identical experimental conditions, using a cell line (U937) representing human monocytes/macrophages. The experiments also aimed at evaluating what effect, if any, cellular stimulation could have on the interactions of nuclear proteins with naturally occurring GC-rich elements in a human genomic DNA. In DNA binding assays, several complexes were formed with the conserved regions identified in multi-species sequence alignment. Furthermore, these regions were active in functional assays. The region containing several matches with 9-mers derived from proximal promoters of human genes was not conserved but formed several complexes with nuclear proteins including Sp1, Egr-1, and an unidentified protein. In addition, this region was active in functional assays and responded to cellular stimulations. Overall, the results of the assays suggest an important role for the sequence context of genomic DNA in protein binding and selection.

Exploring the characteristics of sequence elements in proximal promoters of human genes.

Central to reconstruction of cis-regulatory networks is identification and classification of naturally occurring transcription factor-binding sites according to the genes that they control. We have examined salient characteristics of 9-mers that occur in various orders and combinations in the proximal promoters of human genes. In evaluations of a dataset derived with respect to experimentally defined transcription initiation sites, in some cases we observed a clear correspondence of highly ranked 9-mers with protein-binding sites in genomic DNA. Evaluations of the larger dataset, derived with respect to the 5' end of human ESTs, revealed that a subset of the highly ranked 9-mers corresponded to sites for several known transcription factor families (including CREB, ETS, EGR-1, SP1, KLF, MAZ, HIF-1, and STATs) that play important roles in the regulation of vertebrate genes. We identified several highly ranked CpG-containing 9-mers, defining sites for interactions with the CREB and ETS families of proteins, and identified potential target genes for these proteins. The results of the studies imply that the CpG-containing transcription factor-binding sites regulate the expression of genes with important roles in pathways leading to cell-type-specific gene expression and pathways controlled by the complex networks of signaling systems.

Organization of the promoter region of the human NF-IL6 gene.

In monocyte/macrophages, the human NF-IL6 gene was activated by LPS or PMA. However, a robust response required stimulation of cells with both LPS and PMA. To examine the molecular basis of this response, we isolated human genomic DNA and determined the nucleotide sequence of a segment (6.4 kb) that included the transcription initiation site of the gene. The unique sequences in the 6.4-kb DNA include several potential transcription factor-binding elements that may explain the molecular basis of the activation of the human NF-IL6 gene by signaling molecules that control the immune and inflammatory responses. Deletion analysis localized an LPS+PMA responsive region downstream position -287, with respect to the transcription initiation site of the NF-IL6 gene. The responsive region includes a potential site for interactions with CREB and a region (-287 to -247) that interacts with SP1 and SP3. In functional assays, the potential CREB site responded to cellular stimulation. The region that interacted with SP1 and SP3 augmented the overall level of activity produced in response to LPS+PMA.