DNA sequence information underpins genetic research, enabling discoveries of important biological or medical benefit. Sequencing projects have traditionally employed long (400–800 bp) reads, but the existence of reference sequences for the human and many other genomes makes it possible to develop new, fast approaches to re-sequencing, whereby shorter reads are compared to a reference to identify intra-species genetic variation. We report an approach that generates several billion bases of accurate nucleotide sequence per experiment at low cost. Single molecules of DNA are attached to a flat surface, amplified in situ and used as templates for synthetic sequencing with fluorescent reversible terminator deoxyribonucleotides. Images of the surface are analysed to generate high quality sequence. We demonstrate application of this approach to human genome sequencing on flow-sorted X chromosomes and then scale the approach to determine the genome sequence of a male Yoruba from Ibadan, Nigeria. We build an accurate consensus sequence from >30x average depth of paired 35-base reads. We characterise four million SNPs and four hundred thousand structural variants, many of which are previously unknown. Our approach is effective for accurate, rapid and economical whole genome re-sequencing and many other biomedical applications.

The maleimide motif is widely used for the selective chemical modification of cysteine residues in proteins. Despite widespread utilization, there are some potential limitations, including the irreversible nature of the reaction and, hence, the modification and the number of attachment positions. We conceived of a new class of maleimide which would address some of these limitations and provide new opportunities for protein modification. We report herein the use of mono- and dibromomaleimides for reversible cysteine modification and illustrate this on the SH2 domain of the Grb2 adaptor protein (L111C). After initial modification of a protein with a bromo- or dibromomaleimide, it is possible to add an equivalent of a second thiol to give further bioconjugation, demonstrating that bromomaleimides offer opportunities for up to three points of attachment. The resultant protein−maleimide products can be cleaved to regenerate the unmodified protein by addition of a phosphine or a large excess of a thiol. Furthermore, dibromomaleimide can insert into a disulfide bond, forming a maleimide bridge, and this is illustrated on the peptide hormone somatostatin. Fluorescein-labeled dibromomaleimide is synthesized and inserted into the disulfide to construct a fluorescent somatostatin analogue. These results highlight the significant potential for this new class of reagents in protein modification.

No abstract

Although recent methods for the engineering of antibody–drug conjugates (ADCs) have gone some way to addressing the challenging issues of ADC construction, significant hurdles still remain. There is clear demand for the construction of novel ADC platforms that offer greater stability, homogeneity and flexibility. Here we describe a significant step towards a platform for next-generation antibody-based therapeutics by providing constructs that combine site-specific modification, exceptional versatility and high stability, with retention of antibody binding and structure post-modification. The relevance of the work in a biological context is also demonstrated in a cytotoxicity assay and a cell internalization study with HER2-positive and -negative breast cancer cell lines.

The reactions of FeCl(dppe)Cp* and Ru(CtCCtCH)L 2 Cp′ with Na[BPh 4 ] and 1,8diazabicyclo[5.4.0]undec-7-ene (dbu; 2 equiv) in a mixed thf/NEt 3 solvent afford {Cp*(dppe)-Fe}(CtCCtC){Ru(PP)Cp′} (PP ) dppe, Cp′ ) Cp*, 7; PP ) (PPh 3 ) 2 , Cp′ ) Cp, 8). Cyclic voltammetry shows that these mixed Fe/Ru complexes undergo sequential loss of up to three electrons, with the mono-and dioxidized species being isolated following chemical oxidation. Computational (DFT) and spectroscopic (IR, NMR, ESR, Mo ¨ssbauer) studies are consistent with a polarized ground-state structure with oxidation leading to the gradual evolution of cumulenic character in the FeC 4 Ru moiety and a greater degree of orbital mixing between the Fe, C, and Ru centers than found in the related heterometallic complex [{Cp*(dppe)-Fe}(CtCCtC){Re(NO)(PPh 3 )Cp*}] n+ ([6] n+ ). In contrast to the two-electron oxidation products derived from the diiron complex {Cp*(dppe)Fe}(CtCCtC){Fe(dppe)Cp*} (1) and iron/rhenium complex 6, the dications [7] 2+ and [8] 2+ feature a dominant contribution from a singlet ground state. Thus, while 6 behaves in a manner closely related to 1, 7 and 8 are more closely related to {Cp(Ph 3 P) 2 Ru}(CtCCtC){Ru(PPh 3 ) 2 Cp} (2) and {Cp*(dppe)Ru}-(CtCCtC){Ru(dppe)Cp*} (3), clearly demonstrating the pronounced role that choice of metal as well as formal electron count can play in tuning the electronic and magnetic properties of this fascinating class of compound.

Highly homogeneous ADCs are generated by the efficient bridging of interchain disulfide bonds in trastuzumab, using next generation maleimides.

Herein we report the use of next generation maleimides (NGMs) for the construction of a potent antibody-drug conjugate (ADC) via functional disulfide bridging. The linker has excellent stability in blood serum and the ADC, armed with monomethyl auristatin E (MMAE), shows excellent potency and cancer cell selectivity in vitro.

Bromopyridazinedione-mediated bioconjugation to a cysteine containing protein and a disulfide containing peptide is reported. These bioconjugates exhibit excellent hydrolytic stability and cleave in an aqueous, thiol-mediated reducing environment.