Bicyclic Engineered Sortase A Performs Transpeptidation under Denaturing Conditions.

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.

Constrained peptides mimic a viral suppressor of RNA silencing.

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.

Environment-Responsive Peptide Dimers Bind and Stabilize Double-Stranded RNA.

Double-stranded RNAs (dsRNA) possess immense potential for biomedical applications. However, their therapeutic utility is limited by low stability and poor cellular uptake. Different strategies have been explored to enhance the stability of dsRNA, including the incorporation of modified nucleotides, and the use of diverse carrier systems. Nevertheless, these have not resulted in a broadly applicable approach thereby preventing the wide-spread application of dsRNA for therapeutic purposes. Herein, we report the design of dimeric stapled peptides based on the RNA-binding protein TAV2b. These dimers are obtained via disulfide formation and mimic the natural TAV2b assembly. They bind and stabilize dsRNA in the presence of serum, protecting it from degradation. In addition, peptide binding also promotes cellular uptake of dsRNA. Importantly, peptide dimers monomerize under reducing conditions which results in a loss of RNA binding. These findings highlight the potential of peptide-based RNA binders for the stabilization and protection of dsRNA, representing an appealing strategy towards the environment-triggered release of RNA. This can broaden the applicability of dsRNA, such as short interfering RNAs (siRNA), for therapeutic applications.

Protein Macrocyclization for Tertiary Structure Stabilization.

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.

Synergistic DNA- and Protein-Based Recognition Promote an RNA-Templated Bio-orthogonal Reaction.

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.

Protein-RNA interactions: structural characteristics and hotspot amino acids.

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).

In Situ Cyclization of Proteins (INCYPRO): Cross-Link Derivatization Modulates Protein Stability.

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.

RNA Structure and Cellular Applications of Fluorescent Light-Up Aptamers.

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.

Covalent bicyclization of protein complexes yields durable quaternary structures.

Proteins are essential biomolecules and central to biotechnological applications. In many cases, assembly into higher-order structures is a prerequisite for protein function. Under conditions relevant for applications, protein integrity is often challenged, resulting in disassembly, aggregation, and loss of function. The stabilization of quaternary structure has proven challenging, particularly for trimeric and higher-order complexes, given the complexity of involved inter- and intramolecular interaction networks. Here, we describe the chemical bicyclization of homotrimeric protein complexes, thereby increasing protein resistance toward thermal and chemical stress. This approach involves the structure-based selection of cross-linking sites, their variation to cysteine, and a subsequent reaction with a triselectrophilic agent to form a protein assembly with bicyclic topology. Besides overall increased stability, we observe resistance toward aggregation and greatly prolonged shelf life. This bicyclization strategy gives rise to unprecedented protein chain topologies and can enable new biotechnological and biomedical applications.

A rapid assay for miRNA maturation by using unmodified pre-miRNA.

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).

Rolling-circle amplification: unshared advantages in miRNA detection.

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.

A double intramolecular cage contraction within a self-assembled metallo-supramolecular bowl.

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.

Detection of microRNA maturation using unmodified pre-microRNA 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.