RNA aptamers directed to discrete functional sites on a single protein structural domain

Abstract

Cellular regulatory networks are organized such that many proteins have few interactions, whereas a few proteins have many. These densely connected protein "hubs" are critical for the system-wide behavior of cells, and the capability of selectively perturbing a subset of interactions at these hubs is invaluable in deciphering and manipulating regulatory mechanisms. SELEX-generated RNA aptamers are proving to be highly effective reagents for inhibiting targeted proteins, but conventional methods generate one or several aptamer clones that usually bind to a single target site most preferred by a nucleic acid ligand. We advance a generalized scheme for isolating aptamers to multiple sites on a target molecule by reducing the ability of the preferred site to select its cognate aptamer. We demonstrate the use of this scheme by generating aptamers directed to discrete functional surfaces of the yeast TATA-binding protein (TBP). Previously we selected "class 1" RNA aptamers that interfere with the TBP's binding to TATA-DNA. By masking TBP with TATA-DNA or an unamplifiable class 1 aptamer, we isolated a new aptamer class, "class 2," that can bind a TBP.DNA complex and is in competition with binding another general transcription factor, TFIIA. Moreover, we show that both of these aptamers inhibit RNA polymerase II-dependent transcription, but analysis of template-bound factors shows they do so in mechanistically distinct and unexpected ways that can be attributed to binding either the DNA or TFIIA recognition sites. These results should spur innovative approaches to modulating other highly connected regulatory proteins.

Fig. 1.

Isolation of aptamers for discrete functional sites on the surface of TATA-binding protein (TBP). (A) A structural model representing the DNA·TBP·TFIIA·TFIIB quaternary complex (taken from ref. 19). TBP in black, and flanking TFIIB, the two subunits of TFIIA in gray are shown as ribbon models; the DNA is represented using a space-filling model. Human TFIIB was modeled with the crystal structure of the yeast tertiary complex. (B) Predicted secondary structure of AptTBP-12 and AptTBP-101, respectively, using mfold developed by Zuker (18). Capital letters represent randomized region, and lowercase letters signify the constant regions. The “true” aptamer moiety defined by mutagenesis is enclosed in the box.

Fig. 2.

AptTBP-12 and AptTBP-101 recognize the DNA site and the TFIIA site, respectively. Shown is an EMSA result using labeled RNA probes with indicated proteins, DNA, and treatments.

Fig. 3.

Mechanistically distinct inhibitory effects by AptTBP-12 and AptTBP-101 on Pol II-dependent in vitro transcription. (A) Both AptTBP-12 and AptTBP-101 inhibit transcription equally effectively. The TATA-binding protein (TBP) concentration in the whole-cell extract is ≈20 nM. The concentration indicated is that of the aptamers. (B) The inhibitory effects of aptamers are reversed by excess TBP. Transcription in whole-cell yeast extracts by using pG5MLT (adenovirus major late promoter with 5X Gal4-binding sites upstream) in the presence of Gal4-VP16. Aptamer (50 nM) was added with template; TBP (500 nM) was added to extract before aptamer and template.

Fig. 4.

Aptamers in different classes affect preinitiation complex assembly in mechanistically distinct ways. Factors bound to the immobilized template were quantified after washing by Western blot analysis with antibodies to TATA-binding protein (TBP), TFIIA, TFIIB, and TFIIE, and a representative blot is shown beneath each graph. (A) The effects of low concentrations of AptTBP-12 and AptTBP-101 on TFIIA and TFIIB association with template. (B) Effects of aptamers on the four general transcription factors at aptamer concentrations that produce saturating or near saturating inhibitory effects. Graphs shown are representative of three independent experiments, and standard errors are shown. Each data set has been normalized to the no-aptamer data point.