The modification of RNA with fluorophores, affinity tags and reactive moieties is of enormous utility for studying RNA localization, structure and dynamics as well as diverse biological phenomena involving RNA as an interacting partner. Here we report a labeling approach in which the RNA of interest—of either synthetic or biological origin—is modified at its 3′-end by a poly(A) polymerase with an azido-derivatized nucleotide. The azide is later on conjugated via copper-catalyzed or strain-promoted azide–alkyne click reaction. Under optimized conditions, a single modified nucleotide of choice (A, C, G, U) containing an azide at the 2′-position can be incorporated site-specifically. We have identified ligases that tolerate the presence of a 2′-azido group at the ligation site. This azide is subsequently reacted with a fluorophore alkyne. With this stepwise approach, we are able to achieve site-specific, internal backbone-labeling of de novo synthesized RNA molecules.

Despite its key role in Alzheimer pathogenesis, the physiological function(s) of the amyloid precursor protein (APP) and its proteolytic fragments are still poorly understood. Previously, we generated APPsa knock-in (KI) mice expressing solely the secreted ectodomain APPsa. Here, we generated double mutants (APPsa-DM) by crossing APPsa-KI mice onto an APLP2-deficient background and show that APPsa rescues the postnatal lethality of the majority of APP/APLP2 double knockout mice. Surviving APPsa-DM mice exhibited impaired neuromuscular transmission, with reductions in quantal content, readily releasable pool, and ability to sustain vesicle release that resulted in muscular weakness. We show that these defects may be due to loss of an APP/Mint2/Munc18 complex. Moreover, APPsa-DM muscle showed fragmented postsynaptic specializations, suggesting impaired postnatal synaptic maturation and/or maintenance. Despite normal CNS morphology and unaltered basal synaptic transmission, young APPsa-DM mice already showed pronounced hippocampal dysfunction, impaired spatial learning and a deficit in LTP that could be rescued by GABA A receptor inhibition. Collectively, our data show that APLP2 and APP are synergistically required to mediate neuromuscular transmission, spatial learning and synaptic plasticity.

Bioorthogonal reactions are of high interest in biosciences as they allow the introduction of fluorescent dyes, affinity tags, or other unnatural moieties into biomolecules. The site-specific attachment of two or more different labels is particularly demanding and typically requires laborious multistep syntheses. Here, we report that the most popular cycloaddition in bioconjugation, the copper-catalyzed azide-alkyne click reaction (CuAAC), is fully orthogonal to the inverse electron-demand Diels-Alder reaction (DAinv). We demonstrate that both bioorthogonal reactions can be conducted concurrently in a one-pot reaction by just mixing all components. Orthogonality has been established even for highly reactive trans-cyclooctene-based dienophiles (with rate constants up to 380 000 M(-1) s(-1)). These properties allow for the convenient site-specific one-step preparation of oligonucleotide FRET probes and related reporters needed in cellular biology and biophysical chemistry.

In this study, we derivatized type I collagen without altering its triple helical conformation to allow for facile hydrogel formation via Micheal addition of thiols to methacrylates without the addition of other crosslinking agents. This method provides the flexibility needed for fabrication of injectable hydrogels or pre-fabricated implantable scaffolds, using the same components by tuning the modulus from Pa to kPa. Enzymatic degradability of the hydrogels can be also easily fine tuned by variation of the ratio and type of cross-linking component. The structural morphology reveals lamellar structure mimicking native collagen fibrils. The versatility of this material is demonstrated by its use as a pre-fabricated substrate for culturing human corneal epithelial cells, and as an injectable hydrogel for 3-D encapsulation of cardiac progenitor cells.Keywords: bio-orthogonal chemistry, tissue engineering, pre-fabricated scaffolds, injectable scaffolds, cell compatible. IntroductionThe extracellular matrix (ECM) provides mechanical support as well as instructive signals for cell development, migration, proliferation, survival and function. Natural biopolymers derived from the ECM are therefore by nature, very biocompatible and bio-interactive. [1][2][3] The most abundant is collagen that has extensively been used to prepare scaffolds for tissue repair and engineering. This structural ECM component has, however limited number of functional groups that can be used for direct crosslinking. 4, 5 The main functional groups are amine and carboxylic acids, which allows collagen to crosslinked (e.g. via UV, thermal heating, carbodiimides, epoxy or aldehyde crosslinkers). Conversely, synthetic polymers such as poly (ethylene) glycols, poly (lactic acids), poly (methacryl/acryl amides) etc.), are easily chemically modified for facile processing than collagen.6 However, synthetic polymers have issues with biocompatibility, degradability and do not completely integrate within the host.7-9 Hence, synthetic routes to introduce biocompatible crosslinkable modifications on the protein structure, (reactive moieties), are highly desirable for the development of the next generation of regenerative materials for tissue engineering. Particularly, introducing reactive moieties in collagen would expand its functionality allowing for development of a wider range of scaffolds that can serve as regeneration templates. 10, 11 Several routes to render collagen more processable while retaining its in vitro/ in vivo or clinical bio interactive capacity for promoting tissue regeneration have been explored. [12][13][14] The simplest method is blending collagen with natural or synthetic polymers (such as hyaluronic acid, chitosan, poly(ethylene oxide), polylactic acid, and polyglycolic acid) to fabricate scaffolds. 5, 15 Crosslinked collagen-chitosan hydrogels, which were mechanically stronger than collagen alone, promoted angiogenesis and has been used for islet transplantations in murine models. 16 Li et al.co-polymerized collagen with lam...