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Immunoconjugates are essential tools in diagnostics for the detection and quantification of proteins and in cell biology for the characterization of different cell populations as well as for tracking intracellular pathways. In recent years, antibody-drug conjugates (ADCs) have emerged as promising therapeutics to treat cancer and have moved into the focus of interest of the pharmaceutical industry. Here we describe a conjugation method for the generation of antibody conjugates that relies on the formation of a spontaneous isopeptide bond between two peptide tags referred to as SpyTag and KTag. This reaction is catalyzed by SpyLigase, an engineered cell surface protein obtained from Streptococcus pyogenes. We describe the preparation of SpyLigase by expression from E. coli cells, chemical solid-phase synthesis of the KTag peptide and its coupling to reporter molecules and cytotoxins as well as the transient expression from mammalian cells to produce Spy-tagged antibodies. Furthermore, we describe the purification and analytics of the formed conjugates.

Basierend auf der Kristallstruktur eines naturlichen Proteinsubstrats von mikrobieller Transglutaminase, einem Enzym, das die Vernetzung von Proteinen katalysiert, wurde eine Erkennungssequenz fur ortsspezifische Konjugation rational entworfen. Dieses Strukturmimetikum einer nativen Konjugationsstelle wurde konformativ uber eine intramolekulare Disulfidbrucke fixiert und ermoglichte dadurch die effiziente Konjugation eines Reportermolekuls an den therapeutischen monoklonalen Antikorper Cetuximab, ohne dessen Bindungseigenschaften zu beeintrachtigen.

Spontaneous isopeptide bond formation, a stabilizing posttranslational modification that can be found in gram-positive bacterial cell surface proteins, has previously been used to develop a peptide-peptide ligation technology that enables the polymerization of tagged-proteins catalyzed by SpyLigase. Here we adapted this technology to establish a novel modular antibody labeling approach which is based on isopeptide bond formation between two recognition peptides, SpyTag and KTag. Our labeling strategy allows the attachment of a reporting cargo of interest to an antibody scaffold by fusing it chemically to KTag, available via semi-automated solid-phase peptide synthesis (SPPS), while equipping the antibody with SpyTag. This strategy was successfully used to engineer site-specific antibody-drug conjugates (ADCs) that exhibit cytotoxicities in the subnanomolar range. Our approach may lead to a new class of antibody conjugates based on peptide-tags that have minimal effects on protein structure and function, thus expanding the toolbox of site-specific antibody conjugation.

Orthogonal nucleic acids are chemically modified nucleic acid polymers that are unable to transfer information with natural nucleic acids and thus can be used in synthetic biology to store and transfer genetic information independently. Recently, it was proposed that xylose-DNA (dXNA) can be considered to be a potential candidate for an orthogonal system. Herein, we present the structure in solution and conformational analysis of two self-complementary, fully modified dXNA oligonucleotides, as determined by CD and NMR spectroscopy. These studies are the initial experimental proof of the structural orthogonality of dXNAs. In aqueous solution, dXNA duplexes predominantly form a linear ladderlike (type-1) structure. This is the first example of a furanose nucleic acid that adopts a ladderlike structure. In the presence of salt, an equilibrium exists between two types of duplex form. The corresponding nucleoside triphosphates (dXNTPs) were synthesized and evaluated for their ability to be incorporated into a growing DNA chain by using several natural and mutant DNA polymerases. Despite the structural orthogonality of dXNA, DNA polymerase β mutant is able to incorporate the dXNTPs, showing DNA-dependent dXNA polymerase activity. ispartof: Chemistry - a European Journal vol:18 issue:3 pages:869-879 ispartof: location:Germany status: published

Based on the crystal structure of a natural protein substrate for microbial transglutaminase, an enzyme that catalyzes protein crosslinking, a recognition motif for site-specific conjugation was rationally designed. Conformationally locked by an intramolecular disulfide bond, this structural mimic of a native conjugation site ensured efficient conjugation of a reporter cargo to the therapeutic monoclonal antibody cetuximab without erosion of its binding properties.

Graphical abstract, Modified nucleoside triphosphates (NTPs) represent powerful building blocks to generate nucleic acids with novel properties by enzymatic synthesis. We have recently demonstrated the access to 2′-SeCH3-uridine and 2′-SeCH3-cytidine derivatized RNAs for applications in RNA crystallography, using the corresponding nucleoside triphosphates and distinct mutants of T7 RNA polymerase. In the present note, we introduce the chemical synthesis of the novel 2′-methylseleno-2′-deoxyadenosine and -guanosine 5′-triphosphates (2′-SeCH3-ATP and 2′-SeCH3-GTP) that represent further candidates for the enzymatic RNA synthesis with engineered RNA polymerases.

8-Vinyl- and 8-styryl-2′-deoxyguanosine are synthesized, converted into corresponding 5′-triphosphates and processed by various DNA polymerases to create fluorescent DNA.

Fluorescent analogs of the natural nucleobases are widely used as molecular probes for investigating DNA hybridization and topology. In this study the guanosine analogs 8-vinyl- and 8-styryl-2′-deoxyguanosine were synthesized and converted into the corresponding 5′-triphosphates. These C8 modified nucleotides were processed by various DNA polymerases to create fluorescent DNA. Whereas the 8-styryl modified nucleotide somewhat hampers DNA synthesis 8-vinyl-2′-deoxyguanosine is processed by DNA polymerases emphasizing the broad applicability as a molecular probe for fluorescence spectroscopy.

Selenium-derivatization of RNA is a powerful and advantageous alternative to conventional heavy atom derivatization techniques that are required for the phasing of X-ray crystallographic diffraction data. Among several possibilities, the 2′-methylseleno (2′-SeCH3) modification has been most widely explored and was responsible for a series of important RNA structure determinations, such as the Diels–Alder ribozyme or complexes of antibiotics to HIV dimerization initiation site (DIS) RNA. So far, 2′-SeCH3-RNA has only been accessible by chemical solid-phase synthesis for sizes of up to 50 nucleotides and up to about 100 nucleotides in combination with enzymatic ligation procedures. To overcome this limitation, here we present the enzymatic synthesis of 2′-SeCH3-RNA to open up access for the preparation of long selenium-modified RNA sequences, which cannot be accomplished by conventional chemical synthesis. Therefore, we first elaborated a synthetic route towards the 2′-methylseleno-2′-deoxyribonucleoside triphosphates of cytosine and uridine (2′-SeCH3–CTP and 2′-SeCH3–UTP). With these crucial derivatives in hand, we found that mutants of T7 RNA polymerase are able to incorporate 2′-SeCH3–CMP and 2′-SeCH3–UMP into RNA, while the wild-type polymerase fails to do so. This study demonstrates the efficient enzymatic synthesis of 2′-SeCH3-modified RNA and, thus, provides a thorough foundation for an alternative derivatization strategy in X-ray crystallographic structure analysis of larger RNAs. Such efforts are currently highly requested because of the steadily increasing number of novel non-coding RNAs whose structural features remain to be elucidated.

We present a screening assay based on fluorescence readout for the directed evolution of T7 RNA polymerase variants with acceptance of 2'-modified nucleotides. By using this screening we were able to identify a T7 RNA polymerase mutant with increased acceptance of 2'-methylseleno-2'-deoxyuridine 5'-triphosphate.