A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture

Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided nucleases have gathered considerable excitement as a tool for genome engineering. However, questions remain about the specificity of target site recognition. Cleavage specificity is typically evaluated by low throughput assays (T7 endonuclease I assay, target amplification followed by high-throughput sequencing), which are limited to a subset of potential off-target sites. Here, we used ChIP-seq to examine genome-wide CRISPR binding specificity at gRNA-specific and gRNA-independent sites for two guide RNAs. RNA-guided Cas9 binding was highly specific to the target site while off-target binding occurred at much lower intensities. Cas9-bound regions were highly enriched in NGG sites, a sequence required for target site recognition by Streptococcus pyogenes Cas9. To determine the relationship between Cas9 binding and endonuclease activity, we applied targeted sequence capture, which allowed us to survey 1200 genomic loci simultaneously including potential off-target sites identified by ChIP-seq and by computational prediction. A high frequency of indels was observed at both target sites and one off-target site, while no cleavage activity could be detected at other ChIP-bound regions. Our results confirm the high-specificity of CRISPR endonucleases and demonstrate that sequence capture can be used as a high-throughput genome-wide approach to identify off-target activity.     

Structure-based design of six novel classes of nonpeptide antagonists of the bradykinin B2 receptor

Six classes of nonpeptide bradykinin antagonists were designed using a template derived from structural studies of peptide antagonists. Several compounds from each class were synthesized and assayed for binding to the human bradykinin B2 receptor. Each family showed compounds active at the level of the smallest template peptide; three classes contained compounds with Kd < 8 microM. These results provide diverse leads for a medicinal chemistry-based optimization program.     

An annotated checklist of the orchids of Nepal

An annotated checklist comprised of 458 taxa of orchids known from Nepal is provided, including 104 genera, 437 species, 16 varieties, 3 subspecies and 2 forma and 18 endemic species. In Nepal, orchid species are distributed from 60–5200 m a.s.l. In the checklist, notes on altitudinal ranges, habit, habitat, global distribution, phenology, etc. are presented.     

Atomistic probing of aptameric binding of CD19 outer membrane domain reveals an “aptamer walking” mechanism

We propose an integrated structural approach to search potential aptamer molecules for targeting cancer receptor proteins. We used the outer cellular domain of the B-lymphocyte antigen, CD19, as the target for this study. First, using available protein-aptamer structures deposited in the protein data bank as resources, structural annotation was performed to seek the most probable binding aptamer and its potential initial configuration to the CD19 structure. Using this initial structure, molecular dynamics (MD) simulations were performed for adjustment of the aptamer-binding. During this process, we observed an “aptamer walking” mechanism of the binding of the single-stranded RNA-aptamer to CD19: the aptamer molecule gradually adjusts its configurations and shifts toward favorable binding positions. However, the target molecule CD19 maintained a relatively stable conformation during this process. The interface area between the RNA-aptamer and CD19 increased from less than 8 nm² to over 12 nm² during a 2-μs MD simulation. Using a stable binding pose as the starting structure, we manually mutated the RNA-aptamer to a DNA-aptamer and found that the interface area was further increased to over 16 nm², indicating a stronger affinity compared to the RNA-aptamer. The RNA- and DNA-aptamers and their stable binding-poses to the CD19 molecule may be used as templates in designing potential aptamer molecules that target the B-cell marker molecule CD19 with enhanced specificity and stability.     

Identification of N-terminal extracellular domain determinants in nicotinic acetylcholine receptor (nAChR) α6 subunits that influence effects of wild-type or mutant β3 subunits on function of α6β2*- or α6β4*-nAChR

Despite the apparent function of naturally expressed mammalian α6*-nicotinic acetylcholine receptors (α6*-nAChR; where * indicates the known or possible presence of additional subunits), their functional and heterologous expression has been difficult. Here, we report that coexpression with wild-type β3 subunits abolishes the small amount of function typically seen for all-human or all-mouse α6β4*-nAChR expressed in Xenopus oocytes. However, levels of function and agonist potencies are markedly increased, and there is atropine-sensitive blockade of spontaneous channel opening upon coexpression of α6 and β4 subunits with mutant β3 subunits harboring valine-to-serine mutations at 9'- or 13'-positions. There is no function when α6 and β2 subunits are expressed alone or in the presence of wild-type or mutant β3 subunits. Interestingly, hybrid nAChR containing mouse α6 and human (h) β4 subunits have function potentiated rather than suppressed by coexpression with wild-type hβ3 subunits and potentiated further upon coexpression with hβ3(V9'S) subunits. Studies using nAChR chimeric mouse/human α6 subunits indicated that residues involved in effects seen with hybrid nAChR are located in the α6 subunit N-terminal domain. More specifically, nAChR hα6 subunit residues Asn-143 and Met-145 are important for dominant-negative effects of nAChR hβ3 subunits on hα6hβ4-nAChR function. Asn-143 and additional residues in the N-terminal domain of nAChR hα6 subunits are involved in the gain-of-function effects of nAChR hβ3(V9'S) subunits on α6β2*-nAChR function. These studies illuminate the structural bases for effects of β3 subunits on α6*-nAChR function and suggest that unique subunit interfaces involving the complementary rather than the primary face of α6 subunits are involved.     

Neottia chandrae sp. nov. (Orchidaceae) from Nepal

Neottia chandrae B. B. Raskoti, J. J. Wood & Rita Ale sp. nov. is described and illustrated from Nepal. Notes on the diagnostic characters used to distinguish it from allied species are provided.     

Design,synthesis, and SAR of substituted acrylamides as factor Xa inhibitors

Substituted acrylamides were used as templates that bridge P1 and P4 binding elements, resulting in a series of potent (sub-nanomolar) and selective factor Xa inhibitors. In this template, cis-geometry of P1 and P4 ligands is highly preferred. SAR on the substituting groups, as well as on modification of P1 and P4 moieties is described. Compounds in this series show good in vivo efficacy in animal models.     

Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index

Antibody-drug conjugates enhance the antitumor effects of antibodies and reduce adverse systemic effects of potent cytotoxic drugs. However, conventional drug conjugation strategies yield heterogenous conjugates with relatively narrow therapeutic index (maximum tolerated dose/curative dose). Using leads from our previously described phage display-based method to predict suitable conjugation sites, we engineered cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not perturb immunoglobulin folding and assembly, or alter antigen binding. When conjugated to monomethyl auristatin E, an antibody against the ovarian cancer antigen MUC16 is as efficacious as a conventional conjugate in mouse xenograft models. Moreover, it is tolerated at higher doses in rats and cynomolgus monkeys than the same conjugate prepared by conventional approaches. The favorable in vivo properties of the near-homogenous composition of this conjugate suggest that our strategy offers a general approach to retaining the antitumor efficacy of antibody-drug conjugates, while minimizing their systemic toxicity.     

Essential residues for the enzyme activity of ATP-dependent MurE ligase from Mycobacterium tuberculosis

The emergence of total drug-resistant tuberculosis (TDR-TB) has made the discovery of new therapies for tuberculosis urgent. The cytoplasmic enzymes of peptidoglycan biosynthesis have generated renewed interest as attractive targets for the development of new antimycobacterials. One of the cytoplasmic enzymes, uridine diphosphate (UDP)-MurNAc-tripeptide ligase (MurE), catalyses the addition of meso-diaminopimelic acid (m-DAP) into peptidoglycan in Mycobacterium tuberculosis coupled to the hydrolysis of ATP. Mutants of M. tuberculosis MurE were generated by replacing K157, E220, D392, R451 with alanine and N449 with aspartate, and truncating the first 24 amino acid residues at the N-terminus of the enzyme. Analysis of the specific activity of these proteins suggested that apart from the 24 N-terminal residues, the other mutated residues are essential for catalysis. Variations in K_m values for one or more substrates were observed for all mutants, except the N-terminal truncation mutant, indicating that these residues are involved in binding substrates and form part of the active site structure. These mutant proteins were also tested for their specificity for a wide range of substrates. Interestingly, the mutations K157A, E220A and D392A showed hydrolysis of ATP uncoupled from catalysis. The ATP hydrolysis rate was enhanced by at least partial occupation of the uridine nucleotide dipeptide binding site. This study provides an insight into the residues essential for the catalytic activity and substrate binding of the ATP-dependent MurE ligase. Since ATP-dependent MurE ligase is a novel drug target, the understanding of its function may lead to development of novel inhibitors against resistant forms of M. tuberculosis.