Naturally occurring three-way junctions can be repurposed as genetically encoded RNA-based sensors.

Small molecules can be imaged in living cells using biosensors composed of RNA. However, RNA-based devices are difficult to design. Here, we describe a versatile platform for designing RNA-based fluorescent small-molecule sensors using naturally occurring highly stable three-way junction RNAs. We show that ligand-binding aptamers and fluorogenic aptamers can be inserted into three-way junctions and connected in a way that enables the three-way junction to function as a small-molecule-regulated fluorescent sensor in vitro and in cells. The sensors are designed so that the interhelical stabilizing interactions in the three-way junction are only induced upon ligand binding. We use these RNA-based devices to measure the dynamics of S-adenosylmethionine levels in mammalian cells in real time. We show that this strategy is compatible with diverse metabolite-binding RNA aptamers, fluorogenic aptamers, and three-way junctions. Overall, these data demonstrate a versatile method for readily generating RNA devices that function in living cells.

RIBOTACs: Small Molecules Target RNA for Degradation.

Selective RNA degradation is a powerful strategy to combat diseases or study specific RNA function. In this issue of Cell Chemical Biology, Costales et al. (2019) develop a small molecule that mediates selective RNA degradation with the potential for cancer therapeutics.

Accessibility of Densely Localized DNA on Soft Polymer Nanoparticles.

The dense localization of DNA on soluble nanoparticles can lead to effects distinct from equivalent amounts of the DNA in solution. However, the specific effect may depend on the nature of the assembly and the nanoparticle core. Here we examine the accessibility of densely packed DNA duplexes that extend from a bottle-brush polymer core. We find that unlike spherical nucleic acids, the DNA duplex bristles on the bottle-brush polymer remain accessible to sequence-specific cleavage by endonucleases. In addition, the hybridized strand of the duplex can be displaced through a toehold-mediated strand exchange even at the polymer interface. These results demonstrate that the DNA on bottle-brush polymer remains sufficiently flexible to allow enzymatic degradation or DNA hybridization.

Eliminating Spurious Zero-Efficiency FRET States in Diffusion-Based Single-Molecule Confocal Microscopy.

Single-molecule Förster resonance energy transfer (smFRET) of freely diffusing biomolecules using confocal microscopy is a simple and powerful technique for measuring conformation and dynamics. However, a spurious zero-FRET population can significantly distort the measured histograms and lead to incorrect results, particularly in measurements of intrinsically low-FRET systems. Using a model system consisting of duplex DNAs, we show that there are two important contributions to the zero-FRET state: (1) formation of a dark triplet state of the acceptor dye and (2) the presence of donor-only strands due to incomplete hybridization between donor- and acceptor-labeled strands. The combined strategy of using Trolox as a triplet-state quencher and labeling the same DNA strand with donor and acceptor dyes effectively eliminates the zero-FRET population, even for constructs with intrinsically low FRET efficiencies. This strategy allows us to perform smFRET experiments using a simple confocal microscope with improved accuracy.

Crystal structure of the Entamoeba histolytica RNA lariat debranching enzyme EhDbr1 reveals a catalytic Zn2+ /Mn2+ heterobinucleation.

The RNA lariat debranching enzyme, Dbr1, is a metallophosphoesterase that cleaves 2'-5' phosphodiester bonds within intronic lariats. Previous reports have indicated that Dbr1 enzymatic activity is supported by diverse metal ions including Ni2+ , Mn2+ , Mg2+ , Fe2+ , and Zn2+ . While in initial structures of the Entamoeba histolytica Dbr1 only one of the two catalytic metal-binding sites were observed to be occupied (with a Mn2+ ion), recent structures determined a Zn2+ /Fe2+ heterobinucleation. We solved a high-resolution X-ray crystal structure (1.8 Å) of the E. histolytica Dbr1 and determined a Zn2+ /Mn2+ occupancy. ICP-AES corroborate this finding, and in vitro debranching assays with fluorescently labeled branched substrates confirm activity.

Pseudo-Ligandless Click Chemistry for Oligonucleotide Conjugation.

Particularly for its use in bioconjugations, the copper-catalyzed (or copper-promoted) azide-alkyne cycloaddition (CuAAC) reaction or 'click chemistry', has become an essential component of the modern chemical biologist's toolbox. Click chemistry has been applied to DNA, and more recently, RNA conjugations, and the protocols presented here can be used for either. The reaction can be carried out in aqueous buffer, and uses acetonitrile as a minor co-solvent that serves as a ligand to stabilize the copper. The method also includes details on the analysis of the reaction product. © 2016 by John Wiley & Sons, Inc.

Solid-phase incorporation of an ATRP initiator for polymer-DNA biohybrids.

The combination of polymers with nucleic acids leads to materials with significantly advanced properties. To obviate the necessity and complexity of conjugating two macromolecules, a polymer initiator is described that can be directly covalently linked to DNA during solid-phase synthesis. Polymer can then be grown from the DNA bound initiator, both in solution after the DNA-initiator is released from the solid support as well as directly on the solid support, simplifying purification. The resulting polymer-DNA hybrids were examined by chromatography and fluorescence methods that attested to the integrity of hybrids and the DNA. The ability to use DNA-based supports expands the range of readily available molecules that can be used with the initiator, as exemplified by direct synthesis of a biotin polymer hybrid on solid-support. This method expands the accessibility and range of advanced polymer biohybrid materials.

Autotransfecting short interfering RNA through facile covalent polymer escorts.

Short interfering ribonucleic acids (siRNAs) are important agents for RNA interference (RNAi) that have proven useful in gene function studies and therapeutic applications. However, the efficacy of exogenous siRNAs for gene knockdown remains hampered by their susceptibility to cellular nucleases and impermeability to cell membranes. We report here new covalent polymer-escort siRNA constructs that address both of these constraints simultaneously. By simple postsynthetic click conjugation of polymers to the passenger strand of an siRNA duplex followed by annealing with the complementary guide strand, we obtained siRNA in which one strand includes terminal polymer escorts. The polymer escorts both confer protection against nucleases and facilitate cellular internalization of the siRNA. These autotransfecting polymer-escort siRNAs are viable in RNAi and effective in knocking down reporter and endogenous genes.