Re-engineered guide RNA enables DNA loops and contacts modulating repression in E. coli.

RNA serves as information media as well as molecular scaffold in nature and synthetic systems. The single guide RNA (sgRNA) widely applied in CRISPR techniques exemplifies both functions, with a guide region bearing DNA base-pairing information, and a structural motif for Cas9 protein scaffolding. The scaffold region has been modified by fusing RNA aptamers to the tetra-stem loop. The guide region is typically not regarded as a pluggable module as it encodes the essential function of DNA sequence recognition. Here, we investigate a chimera of two sgRNAs, with distinct guide sequences joined by an RNA linker (dgRNA), regarding its DNA binding function and loop induction capability. First, we studied the sequence bi-specificity of the dgRNA and discovered that the RNA linker allows distal parts of double-stranded DNA to be brought into proximity. To test the activity of the dgRNA in organisms, we used the LacZ gene as a reporter and recapitulated the loop-mediated gene inhibition by LacI in E. coli. We found that the dgRNA can be applied to target distal genomic regions with comparable levels of inhibition. The capability of dgRNA to induce DNA contacts solely requires dCas9 and RNA, making it a minimal system to remodel chromosomal conformation in various organisms.

A DNA robotic switch with regulated autonomous display of cytotoxic ligand nanopatterns.

The clustering of death receptors (DRs) at the membrane leads to apoptosis. With the goal of treating tumours, multivalent molecular tools that initiate this mechanism have been developed. However, DRs are also ubiquitously expressed in healthy tissue. Here we present a stimuli-responsive robotic switch nanodevice that can autonomously and selectively turn on the display of cytotoxic ligand patterns in tumour microenvironments. We demonstrate a switchable DNA origami that normally hides six ligands but displays them as a hexagonal pattern 10 nm in diameter once under higher acidity. This can effectively cluster DRs and trigger apoptosis of human breast cancer cells at pH 6.5 while remaining inert at pH 7.4. When administered to mice bearing human breast cancer xenografts, this nanodevice decreased tumour growth by up to 70%. The data demonstrate the feasibility and opportunities for developing ligand pattern switches as a path for targeted treatment.

Structure of a mitochondrial ribosome with fragmented rRNA in complex with membrane-targeting elements.

Mitoribosomes of green algae display a great structural divergence from their tracheophyte relatives, with fragmentation of both rRNA and proteins as a defining feature. Here, we report a 2.9 Å resolution structure of the mitoribosome from the alga Polytomella magna harbouring a reduced rRNA split into 13 fragments. We found that the rRNA contains a non-canonical reduced form of the 5S, as well as a permutation of the LSU domain I. The mt-5S rRNA is stabilised by mL40 that is also found in mitoribosomes lacking the 5S, which suggests an evolutionary pathway. Through comparison to other ribosomes with fragmented rRNAs, we observe that the pattern is shared across large evolutionary distances, and between cellular compartments, indicating an evolutionary convergence and supporting the concept of a primordial fragmented ribosome. On the protein level, eleven peripherally associated HEAT-repeat proteins are involved in the binding of 3' rRNA termini, and the structure features a prominent pseudo-trimer of one of them (mL116). Finally, in the exit tunnel, mL128 constricts the tunnel width of the vestibular area, and mL105, a homolog of a membrane targeting component mediates contacts with an inner membrane bound insertase. Together, the structural analysis provides insight into the evolution of the ribosomal machinery in mitochondria.