RESUMEN
Monoclonal antibodies (mAbs) are used extensively as biotherapeutics for chronic and acute conditions. Production of mAbs is lengthy and expensive, with protein A affinity capture the most costly step, due both to the nature of the resin and its marked reduction in binding capacity with repeated use. Our previous studies using in situ ATR-FTIR spectroscopy indicated that loss in protein A binding capacity is not the result of leaching or degradation of protein A ligand, suggesting fouling is the principal cause. Here we explore binding behavior and resin capacity loss using Raman spectroscopy. Our data reveal a distinct Raman spectral fingerprint for mAb bound to the protein A ligand of MabSelect SuRe. The results show that the drop in static binding capacity (SBC) previously observed for used protein A resin is discernible by Raman spectroscopy in combination with partial least-squares regression. The SBC is lowest (35.76 mg mL-1) for used inlet resin compared to used outlet (40.17 mg mL-1) and unused resin samples (70.35 mg mL-1). Depth profiling by Raman spectroscopy indicates that at below saturating concentrations (â¼18 mg mL-1), binding of mAb is not homogeneous through used resin beads with protein binding preferentially to the outer regions of the bead, in contrast to fully homogeneous distribution through unused control MabSelect SuRe resin beads. Analysis of the Raman spectra indicates that one foulant is irreversibly bound mAb. The presence of irreversibly bound mAb and host cell proteins was confirmed by mass spectrometric analysis of used resin beads.
Asunto(s)
Espectrometría Raman , Proteína Estafilocócica A , Proteína Estafilocócica A/química , Ligandos , Cromatografía de Afinidad/métodos , Anticuerpos Monoclonales/químicaRESUMEN
Protein conjugates are valuable tools for studying biological processes or producing therapeutics, such as antibody-drug conjugates. Despite the development of several protein conjugation strategies in recent years, the ability to modify one specific amino acid residue on a protein in the presence of other reactive side chains remains a challenge. We show that monosubstituted cyclopropenone (CPO) reagents react selectively with the 1,2-aminothiol groups of N-terminal cysteine residues to give a stable 1,4-thiazepan-5-one linkage under mild, biocompatible conditions. The CPO-based reagents, all accessible from a common activated ester CPO-pentafluorophenol (CPO-PFP), allow selective modification of N-terminal cysteine-containing peptides and proteins even in the presence of internal, solvent-exposed cysteine residues. This approach enabled the preparation of a dual protein conjugate of 2×cys-GFP, containing both internal and N-terminal cysteine residues, by first modifying the N-terminal residue with a CPO-based reagent followed by modification of the internal cysteine with a traditional cysteine-modifying reagent. CPO-based reagents enabled a copper-free click reaction between two proteins, producing a dimer of a de novo protein mimic of IL2 that binds to the ß-IL2 receptor with low nanomolar affinity. Importantly, the reagents are compatible with the common reducing agent dithiothreitol (DTT), a useful property for working with proteins prone to dimerization. Finally, quantum mechanical calculations uncover the origin of selectivity for CPO-based reagents for N-terminal cysteine residues. The ability to distinguish and specifically target N-terminal cysteine residues on proteins facilitates the construction of elaborate multilabeled bioconjugates with minimal protein engineering.
Asunto(s)
Cisteína , Proteínas , Ciclopropanos , Cisteína/química , Indicadores y Reactivos , Proteínas/químicaRESUMEN
5-(Alkynyl)dibenzothiophenium triflates are introduced as new reagents to prepare different protein conjugates through site-selective cysteine alkynylation. The protocol developed allows a highly efficient label of free cysteine-containing proteins with relevant biological roles, such as ubiquitin, the C2A domain of Synaptotagmin-I, or HER2 targeting nanobodies. An electrophilic bis-alkynylating reagent was also designed. The second alkynylating handle thus introduced in the desired protein enables access to protein-thiol, protein-peptide, and protein-protein conjugates, and even diubiquitin dimers can be prepared through this approach. The low excess of reagent needed, mild reaction conditions used, short reaction times, and stability of the S-C(alkyne) bonds at physiological conditions make this approach an interesting addition to the toolbox of classical, site-selective cysteine-conjugation methods.
Asunto(s)
Alquinos/química , Proteínas/química , Tiofenos/química , Alquinos/síntesis química , Animales , Técnicas de Química Sintética , Cisteína/síntesis química , Cisteína/química , Humanos , Indicadores y Reactivos , Mesilatos/síntesis química , Mesilatos/química , Modelos Moleculares , Proteínas/síntesis química , Compuestos de Sulfhidrilo/química , Tiofenos/síntesis químicaRESUMEN
This Communication reports the first general method for rapid, chemoselective, and modular functionalization of serine residues in native polypeptides, which uses a reagent platform based on the P(V) oxidation state. This redox-economical approach can be used to append nearly any kind of cargo onto serine, generating a stable, benign, and hydrophilic phosphorothioate linkage. The method tolerates all other known nucleophilic functional groups of naturally occurring proteinogenic amino acids. A variety of applications can be envisaged by this expansion of the toolbox of site-selective bioconjugation methods.
Asunto(s)
Péptidos/química , Serina/química , Secuencia de Aminoácidos , Aminoácidos/química , Sitios de Unión , Modelos Moleculares , Oxidación-Reducción , Oligonucleótidos Fosforotioatos/química , Fosforilación , Conformación Proteica , Ubiquitina/químicaRESUMEN
An azanorbornadiene bromovinyl sulfone reagent for cysteine-selective bioconjugation has been developed. Subsequent reaction with dipyridyl tetrazine leads to bond cleavage and formation of a pyrrole-linked conjugate. The latter involves ligation of the tetrazine to the azanorbornadiene-tagged protein through inverse electron demand Diels-Alder cycloaddition with subsequent double retro-Diels-Alder reactions to form a stable pyrrole linkage. The sequence of site-selective bioconjugation followed by bioorthogonal bond cleavage was efficiently employed for the labelling of three different proteins. This method benefits from easy preparation of these reagents, selectivity for cysteine, and stability after reaction with a commercial tetrazine, which has potential for the routine preparation of protein conjugates for chemical biology studies.
Asunto(s)
Compuestos Aza/química , Norbornanos/química , Pirroles/química , Reacción de Cicloadición , Cisteína/químicaRESUMEN
Tc-DNA is a conformationally constrained oligonucleotide analogue which shows significant increase in thermal stability when hybridized with RNA, DNA or tc-DNA. Remarkably, recent studies revealed that tc-DNA antisense oligonucleotides (AO) hold great promise for the treatment of Duchenne muscular dystrophy and spinal muscular atrophy. To date, no high-resolution structural data is available for fully modified tc-DNA duplexes and little is known about the origins of their enhanced thermal stability. Here, we report the structures of a fully modified tc-DNA oligonucleotide paired with either complementary RNA, DNA or tc-DNA. All three investigated duplexes maintain a right-handed helical structure with Watson-Crick base pairing and overall geometry intermediate between A- and B-type, but closer to A-type structures. All sugars of the tc-DNA and RNA residues adopt a North conformation whereas the DNA deoxyribose are found in a South-East-North conformation equilibrium. The conformation of the tc-DNA strand in the three determined structures is nearly identical and despite the different nature and local geometry of the complementary strand, the overall structures of the examined duplexes are very similar suggesting that the tc-DNA strand dominates the duplex structure.
Asunto(s)
ADN/química , Conformación de Ácido Nucleico , Oligonucleótidos/química , ARN/química , Emparejamiento Base , Dicroismo Circular , ADN/genética , Desoxirribosa , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Hibridación de Ácido Nucleico , Oligonucleótidos/genética , ARN/genética , TermodinámicaRESUMEN
We describe the synthesis, thermal stability, structural and RNase H activation properties of 2'ß-fluoro-tricyclo nucleic acids (2'F-tc-ANA). Three 2'F-tc-ANA nucleosides (T, 5Me C and A) were synthesized starting from a previously described fluorinated tricyclo sugar intermediate. NMR analysis and quantum mechanical calculations indicate that 2'F-tc-ANA nucleosides prefer sugar conformations in the East and South regions of the pseudorotational cycle. UV-melting experiments revealed that non-consecutive insertions of 2'F-tc-ANA units in DNA reduce the affinity to DNA and RNA complements. However, an oligonucleotide with five contiguous 2'F-tc-ANA-T insertions exhibits increased affinity to complementary RNA. Moreover, a fully modified 10-mer 2'F-tc-ANA oligonucleotide paired to both DNA (+1.6 °C/mod) and RNA (+2.5 °C/mod) with significantly higher affinity compared to corresponding unmodified DNA, and similar affinity compared to corresponding tc-DNA. In addition, CD spectroscopy and molecular dynamics simulations indicate that the conformation of the 2'F-tc-ANA/RNA duplex is similar to that of a DNA/RNA duplex. Moreover, in some sequence contexts, 2'F-tc-ANA promotes RNase H-mediated cleavage of a complementary RNA strand. Taken together, 2'F-tc-ANA represents a nucleic acid analogue that offers the advantage of high RNA affinity while maintaining the ability to activate RNase H, and can be considered a prospective candidate for gene silencing applications.
Asunto(s)
Ácidos Nucleicos/química , Oligonucleótidos/química , Ribonucleasa H/química , Secuencia de Bases , Conformación de Carbohidratos , ADN/química , Activación Enzimática , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Nucleósidos/química , Oligonucleótidos/síntesis química , Transición de Fase , Teoría Cuántica , ARN/química , TemperaturaRESUMEN
Tricyclo-DNA (tcDNA) is a sugar-modified analogue of DNA currently tested for the treatment of Duchenne muscular dystrophy in an antisense approach. Tandem mass spectrometry plays a key role in modern medical diagnostics and has become a widespread technique for the structure elucidation and quantification of antisense oligonucleotides. Herein, mechanistic aspects of the fragmentation of tcDNA are discussed, which lay the basis for reliable sequencing and quantification of the antisense oligonucleotide. Excellent selectivity of tcDNA for complementary RNA is demonstrated in direct competition experiments. Moreover, the kinetic stability and fragmentation pattern of matched and mismatched tcDNA heteroduplexes were investigated and compared with non-modified DNA and RNA duplexes. Although the separation of the constituting strands is the entropy-favored fragmentation pathway of all nucleic acid duplexes, it was found to be only a minor pathway of tcDNA duplexes. The modified hybrid duplexes preferentially undergo neutral base loss and backbone cleavage. This difference is due to the low activation entropy for the strand dissociation of modified duplexes that arises from the conformational constraint of the tc-sugar-moiety. The low activation entropy results in a relatively high free activation enthalpy for the dissociation comparable to the free activation enthalpy of the alternative reaction pathway, the release of a nucleobase. The gas-phase behavior of tcDNA duplexes illustrates the impact of the activation entropy on the fragmentation kinetics and suggests that tandem mass spectrometric experiments are not suited to determine the relative stability of different types of nucleic acid duplexes. Graphical Abstract á .
Asunto(s)
ADN/química , Conformación de Ácido Nucleico , Entropía , Cinética , ARN , TermodinámicaRESUMEN
The synthesis of 2'-fluoro tricyclo-DNA pyrimidine nucleosides with fluorine in the ribo-configuration and their incorporation into oligodeoxynucleotides was accomplished. Unlike the parent tc-T nucleoside, the 2'F-RNA-tc-T unit occurs in the 2'-exo conformation in the crystal. Specifically, F-RNA-tc-T was found to stabilize duplexes with RNA by +2 to +4 °C in Tm/mod. F-RNA-tc-nucleosides mix well with the DNA backbone and thus open up possibilities of using shorter and mixed-(DNA/tc-DNA) backbone oligonucleotides for therapeutic applications.
Asunto(s)
ADN/química , Flúor/química , Furanos/química , ARN/química , Modelos Moleculares , Conformación Molecular , Pirimidinas/químicaRESUMEN
The synthesis of the two fluorinated tricyclic nucleosides 6'-F-tc-T and 6'-F-tc-5(Me)C, as well as the corresponding building blocks for oligonucleotide assembly, was accomplished. An X-ray analysis of N(4)-benzoylated 6'-F-tc-(5Me)C reavealed a 2'-exo (north) conformation of the furanose ring, characterizing it as an RNA mimic. In contrast to observations in the bicyclo-DNA series, no short contact between the fluorine atom and the H6 of the base, reminiscent of a nonclassical F···H hydrogen bond, could be observed. Tm measurements of modified oligodeoxynucleotides with complementary RNA showed slightly sequence-dependent duplex stabilization profiles with maximum ΔTm/mod values of +4.5 °C for 6'-F-tc-(5Me)C and +1 °C for 6'-F-tc-T. In comparison with parent tc-modified oligonucleotides, no relevant changes in Tm were detected, attributing the fluorine substituent a neutral role in RNA affinity. A structural analysis of duplexes with DNA and RNA by CD-spectroscopy revealed a shift from B- to A-type conformation induced by the 6'-F-tc-nucleosides. This is not a specific "fluorine effect", as the same is also observed for the parent tc-modifications. The two fluorinated tc-nucleosides were also incorporated into a pure tricyclo-DNA backbone and showed no discrimination in Tm with complementary RNA, demonstrating that 6'-F substitution is also compatible within fully modified tc-oligonucleotides.