Project

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Chemical biology in microRNA pathways

Applicant Hall Jonathan
Number 124720
Funding scheme Project funding (Div. I-III)
Research institution Institut für Pharmazeutische Wissenschaften ETH Zürich
Institution of higher education ETH Zurich - ETHZ
Main discipline Organic Chemistry
Start/End 01.01.2010 - 31.12.2012
Approved amount 380'550.00
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All Disciplines (2)

Discipline
Organic Chemistry
Biochemistry

Keywords (8)

miRNA; microRNA; antisense; miR-122; RNA hairpin; pre-miRNA; antisense oligonucleotide; antagomir

Lay Summary (English)

Lead
Lay summary
In recent years a new class of small cellular ribonucleic acids (RNA) has been discovered in man, and many other species. Emerging data shows that these RNAs - microRNAs (miRNAs) - may play important roles in disease. Research to clarify their roles however has been hindered by their complex biology, in particular by the poorly understood way in which they interact with their partner messenger RNAs in cells. New chemical tools are needed to help elucidate this biology. A general objective of this research project is to contribute new chemistry and chemical technologies, to be used in combination with bioinformatics and genetics approaches, to help clarify the function of cellular miRNAs. This will lead to the identification of those miRNAs which play crucial roles in disease mechanisms and thereby highlight them as potential drug targets. Historically, it has been difficult to develop classical drugs which bind RNA targets effectively. One class of drug molecules however, antisense oligonucleotides (ASO) bind target RNAs predictably, strongly and selectively but are difficult to use in man, partly because of the large size of the compounds. A specific objective of the project plan is to reduce the size of such oligonucleotides through two avenues of research: chemically-modification of the ASOs and indirectly, by new targeting strategies. A number of novel chemically-modified oligonucleotides will be synthesized and tested for improved properties in human cancer cells. Targeting well-characterised precursor molecules of miRNAs is an alternative strategy to improving drug properties. We will test both concepts using the human miRNA miR-122 which plays an important but poorly-understood role in the replication of hepatitis C viral RNA in liver. The research will bring an increased understanding of the factors which govern strong interactions between drug molecules and miRNA structures and lessons will be applicable to other structurally-similar RNA species in cells.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Structural basis of pre-let-7 miRNA recognition by the zinc knuckles of pluripotency factor Lin28
Loughlin Fionna E. Gebert Luca F.R. Towbin Harry Brunschweiger Andreas Hall Jonathan Allain Fr (2011), Structural basis of pre-let-7 miRNA recognition by the zinc knuckles of pluripotency factor Lin28, in Nature structural & molecular biology , 19 (1 ), 84 -89.
Properties of N(4)-methylated cytidines in miRNA mimics.
Guennewig B Stoltz M Menzi M Dogar AM Hall J., Properties of N(4)-methylated cytidines in miRNA mimics., in Nucleic Acid Therapeutics, 22(2), 109-116.
Systematic screens of proteins binding to synthetic microRNA precursors
Harry Towbin Philipp Wenter Boris Guennewig Jochen Imig Julian A. Zagalak André P. Gerber, Systematic screens of proteins binding to synthetic microRNA precursors, in Nucleic Acids Research, 41(3), e47.

Collaboration

Group / person Country
Types of collaboration
University Hospital Zürich Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
ETH Zurich Switzerland (Europe)
- Publication

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
Oligonucleotide Therapeutics Society 02.11.2012 Cambridge, USA
SCS Fall Meeting 13.09.2012 Zürich
SCS fall meeting, (2nd poster) 09.09.2011 Lausanne
SCS fall meeting 09.09.2011 Lausanne
Swiss Chemical Society Meeting 16.09.2010 Zürich
Swiss Chemical Society Meeting (2nd poster) 16.09.2010 Zürich


Associated projects

Number Title Start Funding scheme
144123 Ligands for the modulation of microRNA biogenesis and function 01.01.2013 Project funding (Div. I-III)

Abstract

RNA is a biomolecule with a unique versatility in biomedical research. mRNAs are targets for drugs, synthetic RNAs are biological tools and modified RNAs are being tested as drugs in the clinic. This proposal considers RNA in each of these roles.Emerging data shows that microRNAs (miRNAs) play important roles in pathways associated with disease. In cases where the dysfunction of a miRNA, or a miRNA family, contribute to a disease pathology, inhibition of that miRNA may represent a viable therapeutic strategy.RNA is the macromolecular target for few drugs because, with a few exceptions, it has proven difficult to develop low molecular weight compounds to bind the target with high affinity and selectivity. On the other hand, RNA is a good target for complementary antisense oligonucleotides (ASO). ASOs bind their target RNAs with high affinity and excellent selectivity, but are difficult to develop as drugs because of poor cellular penetration, insufficient stability, limiting pharmacokinetics (PK) and very high manufacturing costs. Partial solutions have been found for some of these issues which, in many cases, are directly caused by the large size of oligonucleotides. In light of rapid developments in the miRNA field there is a growing need for new and improved approaches to target RNA.A major specific objective of the proposed research is to reduce substantially the lengths of inhibitory oligonucleotides without loss of potency or selectivity, thereby facilitating chemical synthesis / purification, lowering costs and improving PK. We will pursue two avenues of research: new modified ribonucleotides and new RNA-targeting strategies. A general objective of this research is to contribute new chemistry and chemical technology in general to help “functionalize” the miRNA genome in combination with bioinformatics and genetics approaches. A new ribonucleoside modification, C5-ethynyl-pyrimidine-ribonucleotide (C5e-ORN) will be developed which is anticipated to bind RNA with high affinity. We will elaborate synthesis of both uridine and cytidine nucleosides, synthesize them into 2’-O-Me-oligonucleotides and measure binding affinity and mismatch selectivity against complementary RNA. We will use this modification in ASOs to target structures in microRNA biogenesis and function. To date, inhibition in the miRNA pathway is limited to the use of relatively long antagomirs complementary to most of the mature miRNA. We will explore several points of intervention including the mature miRNA, as miRNA mimics if the modification is accepted by proteins of the miRNA pathway and as target protectors binding to the mRNA. A special effort will be devoted to binding precursor miRNA loops to inhibit processing. Hairpin loops are often functionally-important structural motifs in RNAs which bind regulatory proteins or non-coding RNAs. Data in the literature suggest that segments of hairpin loops in RNAs are preorganized to bind relatively short complementary oligonucleotides with high affinity. We will investigate the hairpin loop of microRNA precursors as high affinity binding sites for short RNA-based oligonucleotides. With high affinity ligands bound close to the hairpin loop of miRNAs, we expect to inhibit processing of the miRNA precursor. Our goal will be to achieve potent inhibition using 10-15-nt sequences. We will test concepts using miR-122 as the target miRNA. miR-122 plays an important (but not yet understood) role in replication of hepatitis C viral RNA and in other pathways. We will construct dual luciferase reporter genes carrying a validated miR-122 target, the 3’-UTR of the cationic amino acid transporter gene and use them in cell lines which either express (Huh7), or do not express endogenous miR-122 (HeLa). A surface plasmon resonance method using Biacore apparatus will be established to allow measurement of the binding affinity of ligands to microRNA precursors. An in vitro enzymatic assay will be developed to allow testing of ligands for the inhibition of microRNA processing. The plan involves synthesis of large numbers of oligonucleotides for in vitro assays, including DNA, RNA, modified RNA and long (approximately 60nt), pre-miRNA precursors bearing labels at 5’- and/or 3’terminii. Therefore a strong expertise in RNA chemistry is essential for the proposed research.Experience and understanding of the factors which govern a strong binding affinity between interacting molecules will be applicable to other miRNA species or to functionally important hairpins loops in other species of RNA.
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