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/β-catenin pathway activation, which identifies the mechanisms of differentiation induction.

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-His-d-Nal(2')-NMe-Arg-Trp-Lys]-NH (15) and Ac-Nle-c[Asp-His-d-Nal(2')-NMe-Arg-NMe-Trp-NMe-Lys]-NH (17).

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).

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.

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.