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Enzymes are of central importance to many biotechnological and biomedical applications. However, for many potential applications, the required conditions impede enzyme folding and therefore function. The enzyme Sortase A is a transpeptidase that is widely used to perform bioconjugation reactions with peptides and proteins. Thermal and chemical stress impairs Sortase A activity and prevents its application under harsh conditions, thereby limiting the scope for bioconjugation reactions. Here, we report the stabilization of a previously reported, activity-enhanced Sortase A, which suffered from particularly low thermal stability, using the in situ cyclization of proteins (INCYPRO) approach. After introduction of three spatially aligned solvent-exposed cysteines, a triselectrophilic cross-linker was attached. The resulting bicyclic INCYPRO Sortase A demonstrated activity both at elevated temperature and in the presence of chemical denaturants, conditions under which both wild-type Sortase A and the activity-enhanced version are inactive.

The design of high-affinity, RNA-binding ligands has proven very challenging. This is due to the unique structural properties of RNA, often characterized by polar surfaces and high flexibility. In addition, the frequent lack of well-defined binding pockets complicates the development of small molecule binders. This has triggered the search for alternative scaffolds of intermediate size. Among these, peptide-derived molecules represent appealing entities as they can mimic structural features also present in RNA-binding proteins. However, the application of peptidic RNA-targeting ligands is hampered by a lack of design principles and their inherently low bio-stability. Here, the structure-based design of constrained α-helical peptides derived from the viral suppressor of RNA silencing, TAV2b, is described. We observe that the introduction of two inter-side chain crosslinks provides peptides with increased α-helicity and protease stability. One of these modified peptides (B3) shows high affinity for double-stranded RNA structures including a palindromic siRNA as well as microRNA-21 and its precursor pre-miR-21. Notably, B3 binding to pre-miR-21 inhibits Dicer processing in a biochemical assay. As a further characteristic this peptide also exhibits cellular entry. Our findings show that constrained peptides can efficiently mimic RNA-binding proteins rendering them potentially useful for the design of bioactive RNA-targeting ligands.

Biomolecular assemblies composed of proteins and oligonucleotides play a central role in biological processes. While in nature, oligonucleotides and proteins usually assemble via non‐covalent interactions, synthetic conjugates have been developed which covalently link both modalities. The resulting peptide‐oligonucleotide conjugates have facilitated novel biological applications as well as the design of functional supramolecular systems and materials. However, despite the importance of concerted protein/oligonucleotide recognition in nature, conjugation approaches have barely utilized the synergistic recognition abilities of such complexes. Herein, the structure‐based design of peptide‐DNA conjugates that bind RNA through Watson‐Crick base pairing combined with peptide‐mediated major groove recognition is reported. Two distinct conjugate families with tunable binding characteristics have been designed to adjacently bind a particular RNA sequence. In the resulting ternary complex, their peptide elements are located in proximity, a feature that was used to enable an RNA‐templated click reaction. The introduced structure‐based design approach opens the door to novel functional biomolecular assemblies., DNA, Protein, RNA: The structure‐based design of biomolecular conjugates composed of protein and DNA fragments that synergistically bind RNA via Watson‐Crick base pairing and peptide‐mediated major groove recognition is reported. Two distinct conjugate families with tunable binding characteristics were designed whereby two conjugates can simultaneously assemble on a single RNA scaffold. Through ternary complex formation, the peptide elements of each conjugate are drawn into close proximity, and this feature was exploited to enable an RNA‐templated, double click reaction.

The structure-based design of constrained alpha-helical peptides derived from the viral suppressor of RNA silencing TAV2b is described. We observe that the introduction of two inter-side chain crosslinks provides peptides with increased alpha-helicity and protease stability. One of these modified peptides (B3) shows high affinity for different double-stranded RNA structures including a palindromic siRNA as well as microRNA-21 and its precursor pre-miR-21. Notably, B3 binding to pre-miR-21 inhibits Dicer processing in a biochemical assay. As a further characteristic this peptide also exhibits cellular entry.

The cellular functions of RNA are not limited to their role as blueprints for protein synthesis. In particular, noncoding RNA, such as, snRNAs, lncRNAs, miRNAs, play important roles. With increasing numbers of RNAs being identified, it is well known that the transcriptome outnumbers the proteome by far. This emphasizes the great importance of functional RNA characterization and the need to further develop tools for these investigations, many of which are still in their infancy. Fluorescent light‐up aptamers (FLAPs) are RNA sequences that can bind nontoxic, cell‐permeable small‐molecule fluorogens and enhance their fluorescence over many orders of magnitude upon binding. FLAPs can be encoded on the DNA level using standard molecular biology tools and are subsequently transcribed into RNA by the cellular machinery, so that they can be used as fluorescent RNA tags (FLAP‐tags). In this Minireview, we give a brief overview of the fluorogens that have been developed and their binding RNA aptamers, with a special focus on published crystal structures. A summary of current and future cellular FLAP applications with an emphasis on the study of RNA–RNA and RNA–protein interactions using split‐FLAP and Förster resonance energy transfer (FRET) systems is given.

Structural information about protein–RNA complexes supports the understanding of crucial recognition processes in the cell, and it can allow the development of high affinity ligands to interfere with these processes. In this respect, the identification of amino acid hotspots is particularly important. In contrast to protein–protein interactions, in silico approaches for protein–RNA interactions lag behind in their development. Herein, we report an analysis of available protein–RNA structures. We assembled a data set of 322 crystal and NMR structures and analyzed them regarding interface properties. In addition, we describe a computational alanine-scanning approach which provides interaction scores for interface amino acids, allowing the identification of potential hotspots in protein–RNA interfaces. We have made the computational approach available as an online tool, which allows interaction scores to be calculated for any structure of a protein–RNA complex by uploading atomic coordinates to the PRI HotScore web server (https://pri-hotscore.labs.vu.nl).

Advances in protein engineering have made the industrial use of enzymes feasible, though, a more wide-spread application is hampered by the limited stability of enzymes under often required harsh conditions. We developed a protein stabilization technology that involves the in situ cyclization of proteins (INCYPRO) and facilitates the design and semi-synthesis of multicyclic enzymes. These synthetically enhanced durable enzymes add to the current efforts towards a sustainable and green economy.

Since the launch of ChemistryOpen in 2011, more and more governments are mandating publicly funded research results to be made available in an open-access form; the main idea being that scientific research should be available to the public free of charge to allow creative and innovative research. The Research Councils UK (RCUK) mandated that from April 1st, 2013 all publications resulting from RCUK-funded research should be made open access. As a result, over 20 UK-based universities have opened a Wiley Open Access account thereby ensuring the funding for their open-access articles published with Wiley and Wiley-VCH. In the US, the Fair Access to Science and Technology Research (FASTR) bill, which would require funding agencies to make articles reporting on publicly funded research results freely available, is under ongoing discussion. The EU research and innovation program, Horizon 2020, and governments and agencies in Germany, the Netherlands, France and Belgium, are issuing similar open-access mandates. To comply with these growing government-driven initiatives, authors need to make sure that their research papers are openly accessible, and publishers are responding with a variety of open-access options. As a result, many different publishing models have emerged. Traditional publishing houses offer the option to make selected articles openly accessible in subscription-based journals (hybrid journals), and they have also created new fully open-access journals—typically a fee must be paid and so this is called gold open access, also known as the “author-pays” model. In addition, new open-access publishing houses have been founded, which often cater to a specific institution or society (e.g., eLife or the Beilstein journals). Many universities and institutions have created repositories to make the post-peer-review accepted version accessible after publication (green open access), when permitted by the terms of the publisher license. Unfortunately, so-called “predatory” publishers focusing on high publication numbers and revenue rather than scientific considerations have emerged, and their actions have harmed the term “open access” immensely. This has led to authors feeling insecure about open-access publishing in general. Overall, these different models and players and the increasing pressure from governments to publish open access have led to a very diverse publishing landscape in which a uniform and efficient model for making research accessible to everyone has not yet been found. And, it may well be that the time for a single uniform model has passed. Today, we are faced with a publishing landscape in which everything seems possible.

Roll with it: The quantitative analysis of specific miRNAs from biological samples is very likely to revolutionize diagnostics of human disease. A novel method for miRNA analysis employing rolling-circle amplification (RCA) can homogeneously detect miRNA, even at concentrations as low as 10 fM. The use of T4 RNA ligase 2 (T4 RnL2) at elevated temperatures enables very good discrimination of miRNAs differing by a single nucleotide.

The ever-increasing number of different miRNAs and their association with a vast number of cellular dysfunctions and diseases have initiated several groups to investigate miRNA maturation, which ultimately leads to down regulation of a target messenger RNA (mRNA) and its downstream product. A rapid, convenient, and reliable assay to detect the Dicer-mediated miRNA-maturation step may facilitate research in this field. Here we describe the in vitro detection of the Dicer-mediated miRNA maturation step using unmodified pre-miRNA and branched rolling circle amplification.

The ever-increasing number of different miRNAs and their association with a vast number of cellular dysfunctions and diseases have initiated several groups to investigate miRNA maturation, which ultimately leads to down regulation of a target messenger RNA (mRNA) and its downstream product. A rapid, convenient, and reliable assay to detect the Dicer-mediated miRNA-maturation step may facilitate research in this field. Here we describe the in vitro detection of the Dicer-mediated miRNA maturation step using unmodified pre-miRNA and branched rolling circle amplification.

Without labeling RNA: A new rapid assay for micro-RNA maturation was developed. The assay depends on the cleavage of unmodified pre-miRNAs and the subsequent amplification of the mature miRNA by rolling circle amplification (see accompanying scheme).

In the light of recent progress in RNA biology, the need for molecules that bind to RNA and thus may be suited to manipulating RNA-mediated processes is steadily increasing. We present a very short and efficient synthetic route to alkyne-modified neamine and 2-deoxystreptamine derivatives on a half-gram scale. These derivatives are suitable for constructing a library of potential divalent RNA binders by copper-catalysed 1,3-dipolar cycloaddition to diazides (“click chemistry”). The conjugate dimers thus formed inhibitedDicer-mediated micro-RNA maturation with IC50 values between 0.6 and 15 μM.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)

When isolated in the high vacuum of an ESI-FTICR mass spectrometer, bowl-shaped metallo-supramolecular M(6)L(4) assemblies undergo a surprising and mechanistically interesting intramolecular double cage contraction to yield smaller M(3)L(2) cages.

Proteins possess unique molecular recognition capabilities and enzymatic activities, features that are usually tied to a particular tertiary structure. To make use of proteins for biotechnological and biomedical purposes, it is often required to enforce their tertiary structure in order to ensure sufficient stability under the conditions inherent to the application of interest. The introduction of intramolecular crosslinks has proven efficient in stabilizing native protein folds. Herein, we give an overview of methods that allow the macrocyclization of expressed proteins, discussing involved reaction mechanisms and structural implications., Protein function is often tightly linked to a particular tertiary structure. For many protein applications it is required to stabilize the tertiary structure and prevent unfolding. Here, we summarize strategies that have been used to cyclize expressed proteins aiming for the stabilization of their native fold.

Protein macrocyclization represents a very efficient strategy to increase the stability of protein tertiary structures. Here, we describe a panel of novel C3-symmetric tris-electrophilic agents and their use for the cyclization of proteins. These electrophiles are reacted with a protein domain harboring three solvent-exposed cysteine residues, resulting in the in situ cyclization of the protein (INCYPRO). We observe a clear dependency of cross-linking rates on the electrophilicity. All nine obtained cross-linked protein versions show considerably increased thermal stability (up to 29 °C increased melting temperature) when compared to that of the linear precursor. Most interestingly, the degree of stabilization correlates with the hydrophilicity of the cross-link. These results will support the development of novel cross-linked proteins and enable a more rational design process.