Designer RNA-binding proteins

Ref: 13089
A library of protein modules in which each member will bind specifically to a different three-nucleotide sequence of single-stranded RNA. When these modules are combined, they will be able to be used to target any given specific RNA sequence with high specificity.

Key advantages
  • Ability to target specific RNA sequences
  • Delivery of effector domains to specific RNA sequences

Background

The widespread involvement of RNA in gene regulation and disease is only now beginning to be appreciated. RNA regulates gene expression at many levels.

The splicing of mRNA generates proteomic diversity, and non-coding RNAs are being shown to have a variety of regulatory functions.

In fact, although only 2% of the human genome encodes for proteins, it is now realized that almost all of the genome is transcribed, with the bulk giving rise to non-coding RNAs.

The importance of these species is becoming apparent from the identification of splicing-related diseases and associations of non-coding RNAs with cancer, coronary disease and diabetes. Zinc finger proteins that could deliver effector domains to RNA targets with arbitrary sequences would be powerful tools for the biomedical research community with which to understand human biology.

These proteins could allow us to alter gene splicing events both in cell lines and potentially in live animals, to probe ncRNA function and perhaps ultimately to provide therapeutic interventions.

The invention

X-ray crystallography

X-ray crystallography shows that recognition of an AGGUAA motif by a single ZnF is dominated by side-chain hydrogen bonds to the bases and formation of a guanine-tryptophan-guanine "ladder."

The zinc fingers of the SR-like protein ZRANB2 are single-stranded RNA-binding domains that recognize 5' splice site-like sequences.

The two zinc fingers of ZRANB2 (formerly known as ZNF265) can bind to single-stranded RNA with high sequence specificity. We have determined the structure of one of these fingers bound to its RNA target site (AGGUAA - determined by SELEX).

The structure reveals a new class of RNA-binding domain. This ZnF forms a unique guanine-Trp-guanine aromatic stack, and the core nucleotides (GGU) are recognized by an extensive network of protein side-chain hydrogen bonds.

Also notable are the two-headed hydrogen bonds that are formed between arginine side-chains and the two guanines - these interactions appear to provide strong selection for guanine in those positions.

We are using combainatorial methods to create ZRANB2 zinc-finger variants that recognize different three-nucleotide sequences and assembling tandem repeats of these modules to create entirely new proteins that recognize chosen RNA targets.

Applications

In-cell detection of any chosen RNA species (eg small RNAs, messenger RNAs) in living cells Targeted degradation of any chosen RNA species Targeted changes in translation of chosen RNA species

Principal inventors

  • Professor Joel Mackay

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