Röthlisberger Pascal, Berk Christian, Hall Jonathan (2019), RNA Chemistry for RNA Biology, in CHIMIA International Journal for Chemistry
, 73(5), 368-373.
Hagen Timo, Malinowska Anna L., Lightfoot Helen L., Bigatti Martina, Hall Jonathan (2019), Site-Specific Fluorophore Labeling of Guanosines in RNA G-Quadruplexes, in ACS Omega
, 4(5), 8472-8479.
MalinowskaAnna (2019), Research and Development of Oligonucleotides Targeting microRNAs, in Agrawal Sudhir, Gait Michael J (ed.), Royal Society of Chemistry, Cambridge, 151-180.
Menzi Mirjam, Wild Bettina, Pradère Ugo, Malinowska Anna L., Brunschweiger Andreas, Lightfoot Helen L., Hall Jonathan (2017), Towards Improved Oligonucleotide Therapeutics Through Faster Target Binding Kinetics, in Chemistry - A European Journal
, 23(57), 14221-14230.
Niewoehner Ole, Garcia-Doval Carmela, Rostøl Jakob T., Berk Christian, Schwede Frank, Bigler Laurent, Hall Jonathan, Marraffini Luciano A., Jinek Martin (2017), Type III CRISPR–Cas systems produce cyclic oligoadenylate second messengers, in Nature
, 548(7669), 543-548.
MicroRNAs (miRNAs) are small RNAs that regulate gene expression post-transcriptionally, either fine-tuning expression or acting as phenotypic “switches. Some miRNAs play important roles in diseases and are under clinical investigation as drugs or as drug targets, such as miR-34a and miR-103/107, respectively, for cancer and insulin disorders, respectively. Understanding miRNA function requires that their binding partners be identified. MiRNAs bind target messenger RNAs (mRNAs) mostly in their 3’UTRs, using approx. 7 nucleotides (nt) at their 5’ ends (“seed”). This is considered the canonical mechanism of regulation: it leads to translational repression and/or mRNA decay. A miRNA may bind hundreds to thousands of RNAs in a given cell. Accounts of non-canonical miRNA-RNA interactions, e.g. involving the 3’ends of miRNAs are increasing but base-pairing patterns for these interactions are poorly understood. Thus, there is a need for new methods to identify non-canonical binding interactions, particularly for miRNAs that contribute to programs important for gene expression, or for miRNAs with an important role in pathological mechanisms. Although computational predictions have proven crucial in revealing principles of miRNA-dependent mRNA regulation, the gold standard for the identification of miRNA targets is experimental approaches, in which miRNA binding partners are captured covalently in cells and then validated. These wet-bench-based approaches are still in stages of infancy. The overall objective of this proposal is to provide new chemical tools to capture covalently RNA targets of miRNAs in cells. Identification of a miRNA targetome (all targets) is done by two general capture-based approaches, each with its variants, each with strengths and weak points. In one, all expressed miRNAs and their targets are captured under native conditions in a selected cell type genome-wide fashion; in the other, targets of a single, selected miRNA are captured under non-native conditions. The principal basis for this application was our development of miR-CLIP (miRNA cross-linking and immunoprecipitation), a protocol comprising the design and synthesis of psoralen- and biotin-labeled miRNA probes which mimic native miRNAs in cells and cross-link to their “targetome” when transfected into cells. Whereas a miR-CLIP probe for miR-106a cross-linked efficiently to its target mRNAs in HeLa cells, many other miRNAs were much less efficient cross-linkers, suggesting that the psoralen was not optimally positioned in the probe and that probe sequence was important. The main objective of this proposal is to design and develop a second generation of miR-CLIP probes so that efficient reagents can be obtained for all miRNAs, independent of their sequence. This will provide a resource that permits the targetome of any selected (or in the long term, all) miRNA to be determined, in any transfectable cells. For miR-CLIP probes to be of general use, it is critical that cross-linking occurs from the nucleotide bearing the psoralen to the complementary base in a target. In Part 1 of the proposal we will investigate the cross-linking mechanism for 1st generation miR-CLIP reagents using in vitro and reporter assays. In Part 2 we will investigate alternative modes of conjugating psoralen cross-linkers. In Part 3 we will investigate the use of miRNA-mRNA ligation as a new form of cross-linking. The best performing miR-CLIP reagents will be used to capture the targetomes of ongoing miRNAs of interest in our laboratory.