RNA aptamers as effective protein antagonists in a multicellular organism

Abstract

RNA aptamers selected against proteins can be used to modulate specific protein function. Expression of such reagents in cells and whole organisms could provide a means of dissecting and controlling molecular mechanisms in vivo. We demonstrate that Drosophila B52 protein can be specifically inhibited in vitro and in vivo by a multivalent RNA aptamer. This inhibitory aptamer RNA binds B52 avidly and inhibits B52-stimulated pre-mRNA splicing. It can be expressed in cultured cells and whole animals in a stable form that accumulates up to 10% of total mRNA. It binds B52 in vivo and suppresses all phenotypes caused by B52 overexpression. The strategies presented here should prove general in design and expression of functional and therapeutic RNAs.Multiprotein assemblies drive a variety of highly regulated biological processes. To better understand and control such processes, novel reagents are needed to modulate functions of specific proteins in cells and whole organisms. An ideal reagent would, (i) like an antibody, be made to order specifically for a particular protein, (ii) like a small organic molecule, be able to rapidly target a specific protein domain within cells, and (iii) like a conditional allele, be able to exert its effect in whole organisms, but (iv) also be targetable to specific tissues, cells, or stages of development.Although many strategies exist for inactivating genes or gene products, use of RNA aptamers (1) presents several compelling advantages. Extremely rare RNAs that have high affinity for specific proteins can be selected in vitro (SELEX) (2, 3). Genetically controlled induction of such high affinity RNA aptamers could provide a means of rapidly inactivating a specific domain of a protein and thereby assessing its primary function and mechanism of action in vivo. Alternatively, continuous expression of specific RNA aptamers after stable gene transfer could achieve a long-term alteration in the activity of a target protein. In yeast, the constitutive expression of an aptamer against RNA polymerase II caused cell growth defects under conditions in which the RNA polymerase level was artificially reduced (4). Although this study demonstrated the potential of RNA aptamers as inhibitors of protein function in vivo, several advances are needed to apply this approach to multicellular organisms.Previously, we selected and characterized RNA aptamers to B52, a regulator of RNA splicing in Drosophila melanogaster. B52, also known as SRp55, is a member of the Drosophila SR protein family, a group of nuclear proteins that are essential for pre-mRNA splicing and influence splice site choice (5, 6). B52 contains two RNA recognition motifs in its N-terminal half and a domain rich in serine-arginine dipeptide repeats in its C-terminal half (7). RNA aptamers that bind B52 with high affinity (K d ϭ 20-50 nM) and specificity were selected from a large pool of RNAs (8). The B52 binding sites (BBS) on members of this nonclonally derived family of RNA aptamers not only have a ''conserved'' consensus sequen...

Discriminatory aptamer reveals serum response element transcription regulated by cytohesin-2

“…Few examples of intramer inhibition of biological targets in vivo are known (3,28,29). In these previous studies, efforts were taken to stabilize the intramer or to direct it to the correct cellular compartment.…”

“…Intramers were expressed by vaccinia virus vectors that are difficult to handle, require certain safety precautions, and have to be genetically engineered for each aptamer that will have to be expressed (3,28). In another study, transgenic animals had to be engineered for endogenous aptamer expression (29). An alternative strategy uses transfection of expression plasmids that are translocated into the nucleus so that the expression of the aptamers takes place in the nucleus.…”

Perturbation of Discrete Sites on a Single Protein Domain with RNA Aptamers: Targeting of Different Sides of the TATA-Binding Protein (TBP)

“…2) Multiple aptamers can also be strung together to form multivalent aptamers. 30) The effectiveness of multimeric forms of aptamers was confirmed here by the generation and testing of simple homo-multimeric forms, A-37, V-1, and V-2 aptamers. These showed increased affinity for dTBP.…”

“…lead to higher affinity binding. 30) We postulated that if the binding affinity and total mass of V-aptamers is increased, the triple complex will show a migration pattern distinct from that of the TBP-DNA complex. All dimer and tetramer forms of aptamers bind to dTBP more readily than to the corresponding monomers ( Fig.…”

Yeast genetic selections to optimize RNA decoys for transcription factor NF-κB

“…Previous studies have investigated RNA aptamer modification for optimized in vivo expression, typically by appending rationally designed or in vitro selected sequences that increase aptamer expression, stability, or presentation in cells (2,3,16). In contrast, we now present yeast genetic selections to identify aptamer variants that show improved interaction with the target protein.…”