A protein nanocontainer targeting epithelial cancers: rational engineering, biochemical characterization, drug loading and cell delivery.

The development of drug delivery and imaging tools is a major challenge in human health, in particular in cancer pathologies. This work describes the optimization of a protein nanocontainer, belonging to the lectin protein family, for its use in epithelial cancer diagnosis and treatment. Indeed, it specifically targets a glycosidic marker, the T antigen, which is known to be characteristic of epithelial cancers. Its quaternary structure reveals a large hydrated inner cavity able to transport small therapeutic molecules. Optimization of the nanocontainer by site directed mutagenesis allowed controlling loading and release of confined drugs. Doxorubicin confinement was followed, both theoretically and experimentally, and provided a proof of concept for the use of this nanocontainer as a vectorization system. In OVCAR-3 cells, a human ovarian adenocarcinoma cell line that expresses the T antigen, the drug was observed to be delivered inside late endosomes/lysosomes. These results show that this new type of vectorization and imaging device opens new exciting perspectives in nano-theranostic approaches.

α,ß-D-constrained nucleic acids are strong terminators of thermostable DNA polymerases in polymerase chain reaction.

(S(C5'), R(P)) α,ß-D- Constrained Nucleic Acids (CNA) are dinucleotide building blocks that can feature either B-type torsional angle values or non-canonical values, depending on their 5'C and P absolute stereochemistry. These CNA are modified neither on the nucleobase nor on the sugar structure and therefore represent a new class of nucleotide with specific chemical and structural characteristics. They promote marked bending in a single stranded DNA so as to preorganize it into a loop-like structure, and they have been shown to induce rigidity within oligonucleotides. Following their synthesis, studies performed on CNA have only focused on the constraints that this family of nucleotides introduced into DNA. On the assumption that bending in a DNA template may produce a terminator structure, we investigated whether CNA could be used as a new strong terminator of polymerization in PCR. We therefore assessed the efficiency of CNA as a terminator in PCR, using triethylene glycol phosphate units as a control. Analyses were performed by denaturing gel electrophoresis and several PCR products were further analysed by sequencing. The results showed that the incorporation of only one CNA was always skipped by the polymerases tested. On the other hand, two CNA units always stopped proofreading polymerases, such as Pfu DNA polymerase, as expected for a strong replication terminator. Non-proofreading enzymes, e.g. Taq DNA polymerase, did not recognize this modification as a strong terminator although it was predominantly stopped by this structure. In conclusion, this first functional use of CNA units shows that these modified nucleotides can be used as novel polymerization terminators of proofreading polymerases. Furthermore, our results lead us to propose that CNA and their derivatives could be useful tools for investigating the behaviour of different classes of polymerases.

Conformational exchange is critical for the productivity of an oxidative folding intermediate with buried free cysteines.

Much has been learned about the folding of proteins from comparative studies of the folding of proteins that are related in sequence and structure. Observation of the effects of mutations helps account for sequence-specific properties and large variations in folding rates observed in homologous proteins, which are not explained by structure-derived descriptions. The folding kinetics of variants of a ß-stranded protein, toxin α from Naja nigricollis, depends on the length of their loop lk1. These proteins, named Tox60, Tox61, and Tox62, contain four disulfide bonds. We show that their oxidative refolding pathways are similar. Differences in these pathways are restricted to the last step of the reaction, that is, the closure of the last disulfide. At this step, two species of three-disulfide intermediates are observed: intermediate C lacking the B3 disulfide and intermediate D lacking the B2 disulfide. Surprisingly, D is the most productive intermediate for Tox61 despite the low accessibility of its free cysteines. However, in the case of Tox62, its conversion efficiency drops by 2 orders of magnitude and C becomes the most productive intermediate. NMR was used in order to study the structural dynamics of each of these intermediates. Both three-disulfide intermediates of Tox61 exist in two forms, exchanging on the 1- to 100-ms scale. One of these forms is structurally very close to the native Tox61, whereas the other is always significantly more flexible on a picosecond-to-nanosecond timescale. On the other hand, in the case of Tox62, the three-disulfide intermediates only show a native-like structure. The higher conformational heterogeneity of Tox61 intermediate D allows an increased accessibility of its free cysteines to oxidative agents, which explains its faster native disulfide formation. Thus, residue deletion in loop lk1 probably abrogates stabilizing intramolecular interactions, creates conformational heterogeneity, and increases the folding rate of Tox60 and Tox61 compared to Tox62.