BART-Seq: cost-effective massively parallelized targeted sequencing for genomics, transcriptomics, and single-cell analysis.

We describe a highly sensitive, quantitative, and inexpensive technique for targeted sequencing of transcript cohorts or genomic regions from thousands of bulk samples or single cells in parallel. Multiplexing is based on a simple method that produces extensive matrices of diverse DNA barcodes attached to invariant primer sets, which are all pre-selected and optimized in silico. By applying the matrices in a novel workflow named Barcode Assembly foR Targeted Sequencing (BART-Seq), we analyze developmental states of thousands of single human pluripotent stem cells, either in different maintenance media or upon Wnt/ß-catenin pathway activation, which identifies the mechanisms of differentiation induction. Moreover, we apply BART-Seq to the genetic screening of breast cancer patients and identify BRCA mutations with very high precision. The processing of thousands of samples and dynamic range measurements that outperform global transcriptomics techniques makes BART-Seq first targeted sequencing technique suitable for numerous research applications.

Structural Insights into Selective Ligand-Receptor Interactions Leading to Receptor Inactivation Utilizing Selective Melanocortin 3 Receptor Antagonists.

Systematic N-methylated derivatives of the melanocortin receptor ligand, SHU9119, lead to multiple binding and functional selectivity toward melanocortin receptors. However, the relationship between N-methylation-induced conformational changes in the peptide backbone and side chains and melanocortin receptor selectivity is still unknown. We conducted comprehensive conformational studies in solution of two selective antagonists of the third isoform of the melanocortin receptor (hMC3R), namely, Ac-Nle-c[Asp-NMe-His6-d-Nal(2')7-NMe-Arg8-Trp9-Lys]-NH2 (15) and Ac-Nle-c[Asp-His6-d-Nal(2')7-NMe-Arg8-NMe-Trp9-NMe-Lys]-NH2 (17). It is known that the pharmacophore (His6-DNal7-Arg8-Trp9) of the SHU-9119 peptides occupies a ß II-turn-like region with the turn centered about DNal7-Arg8. The analogues with hMC3R selectivity showed distinct differences in the spatial arrangement of the Trp9 side chains. In addition to our NMR studies, we also carried out molecular-level interaction studies of these two peptides at the homology model of hMC3R. Earlier chimeric human melanocortin 3 receptor studies revealed insights regarding the binding and functional sites of hMC3R selectivity. Upon docking of peptides 15 and 17 to the binding pocket of hMC3R, it was revealed that Arg8 and Trp9 side chains are involved in a majority of the interactions with the receptor. While Arg8 forms polar contacts with D154 and D158 of hMC3R, Trp9 utilizes π-π stacking interactions with F295 and F298, located on the transmembrane domain of hMC3R. It is hypothesized that as the frequency of Trp9-hMC3R interactions decrease, antagonistic activity increases. The absence of any interactions of the N-methyl groups with hMC3R suggests that their primary function is to modulate backbone conformations of the ligands.

Systematic Backbone Conformational Constraints on a Cyclic Melanotropin Ligand Leads to Highly Selective Ligands for Multiple Melanocortin Receptors.

Human melanocortin receptors (hMCRs) have been challenging targets to develop ligands that are explicitly selective for each of their subtypes. To modulate the conformational preferences of the melanocortin ligands and improve the biofunctional agonist/antagonist activities and selectivities, we have applied a backbone N-methylation approach on Ac-Nle-c[Asp-His-D-Nal(2')-Arg-Trp-Lys]-NH2 (Ac-Nle(4)-c[Asp(5),D-Nal(2')(7),Lys(10)]-NH2), a nonselective cyclic peptide antagonist at hMC3R and hMC4R and an agonist at hMC1R and hMC5R. Systematic N-methylated derivatives of Ac-Nle(4)-c[Asp(5),D-Nal(2')(7),Lys(10)]-NH2, with all possible backbone N-methylation combinations, have been synthesized and examined for their binding and functional activities toward melanocortin receptor subtypes 1, 3, 4, and 5 (hMCRs). Several N-methylated analogues are selective and potent agonists or antagonists for hMC1R or hMC5R or have selective antagonist activity for hMC3R. The selective hMC1R ligands show strong binding for human melanoma cells. We have also discovered the first universal antagonist (compound 19) for all subtypes of hMCRs.

The effect of multiple N-methylation on intestinal permeability of cyclic hexapeptides.

Recent progress in peptide synthesis simplified the synthesis of multiple N-methylation of peptides. To evaluate how multiple N-methylation affects the bioavailability of peptides, a poly alanine cyclic hexapeptide library (n = 54), varying in the number of N-methyl (N-Me) groups (1-5 groups) and their position, was synthesized. The peptides were evaluated for their intestinal permeability in vitro using the Caco-2 model. Further evaluation of the transport route of chosen analogues was performed using rat excised viable intestinal tissue, a novel colorimetric liposomal model and the parallel artificial membrane permeability assay (PAMPA). While most members were found to have poor permeability (permeability coefficient, P(app) < 1 x 10⁻6 cm/s, lower than mannitol, the marker for paracellular permeability), 10 analogues were found to have high Caco-2 permeability, (P(app) > 1 x 10⁻5 cm/s, similar to testosterone, a marker of transcellular permeability). No correlation was found between the number of N-methylated groups and the enhanced permeability. However, 9/10 permeable peptides in the Caco-2 model included an N-Me placed adjacently to the D-Ala position. While the exact transport route was not fully characterized, the data suggests a facilitated diffusion. It can be concluded that multiple N-methylation of peptides may improve intestinal permeability, and therefore can be utilized in the design of orally available peptide-based therapeutics.

Multiple N-methylation of MT-II backbone amide bonds leads to melanocortin receptor subtype hMC1R selectivity: pharmacological and conformational studies.

Multiple N-methylation is a novel technology to improve bioavailability of peptides and increase receptor subtype selectivity. This technique has been applied here to the superpotent but nonselective cyclic peptide MT-II. A library of all possible 31 backbone N-methylated derivatives has been synthesized and tested for binding and activation at melanocortin receptor subtypes 1, 3, 4, and 5. It turned out that selectivity is improved with every introduced N-methyl group, resulting in several N-methylated selective and potent agonists for the hMC1R. The most potent of these derivatives is N-methylated on four out of five amide bonds in the cyclic structure. Its solution structure indicates a strongly preferred backbone conformation that resembles other alpha-MSH analogs but possesses much less flexibility and in addition distinct differences in the spatial arrangement of individual amino acid side chains.