Combining SELEX and the yeast three-hybrid system for in vivo selection and classification of RNA aptamers

Abstract

Aptamers are small nucleic acid ligands that bind to their targets with specificity and high affinity. They are generated by a combinatorial technology, known as SELEX. This in vitro approach uses iterative cycles of enrichment and amplification to select binders from nucleic acid libraries of high complexity. Here we combine SELEX with the yeast three-hybrid system in order to select for RNA aptamers with in vivo binding activity. As a target molecule, we chose the RNA recognition motif-containing RNA-binding protein Rrm4 from the corn pathogen Ustilago maydis. Rrm4 is an ELAV-like protein containing three N-terminal RNA recognition motifs (RRMs). It has been implicated in microtubule-dependent RNA transport during pathogenic development. After 11 SELEX cycles, four aptamer classes were identified. These sequences were further screened for their in vivo binding activity applying the yeast three-hybrid system. Of the initial aptamer classes only members of two classes were capable of binding in vivo. Testing representatives of both classes against Rrm4 variants mutated in one of the three RRM domains revealed that these aptamers interacted with the third RRM. Thus, the yeast three-hybrid system is a useful extension to the SELEX protocol for the identification and characterization of aptamers with in vivo binding activity.

FIGURE 1.

Course of the SELEX experiment using recombinant Rrm4^GST as a target. (A) SDS-PAGE analysis of recombinant Rrm4^GST preparations after staining with Coomassie brilliant blue. The full-length fusion protein (111 kDa) and putative degradation products (asterisks) are indicated. (B) Graphical representation of the percentages of bound RNA for different cycles of SELEX. DNA from the 11th cycle (asterisk) was cloned and 49 clones were sequenced.

FIGURE 2.

Classification of in vitro selected aptamer sequences. (A) Four aptamer classes were identified (A to D, relative abundances in parenthesis). The designation of each aptamer is followed by its clone abundance in parentheses (+n, additional n sequences with single-point mutations) and the primary sequence of the randomized region (capital letters). Complementary sequences corresponding to stems 1 and 2 are underlined. Lower-case letters implicated in stem–loop formation indicate sequences derived from the constant primer binding sites. A purine-rich stretch in classes A–C, a conserved CA/G sequence in class A, and four GGN repeats in class D are printed in bold. (B) Secondary structure models for the most abundant member of classes A–D are shown. Models are based on structure predictions using Mfold and sequence similarities to known G-quartet structures (Zuker 2003; Homann et al. 2006). (C) Determination of equilibrium dissociation constants (K_d). Representative binding isotherm for the binding of aptamer B1 to Rrm4^GST. The inset shows a Scatchard plot of the binding data with the slope of the curve representing −1/K_d.

FIGURE 3.

Yeast three-hybrid selection of RNA aptamers in vivo. (A) Schematic representation of the yeast three-hybrid system. For simplicity only one MS2 protein and binding site are indicated (the MS2 protein binds as a dimer and the RNA hybrid contains two MS2 binding sites). The label “selex” symbolizes the presence of sequences from the 11th cycle of SELEX. CP, AD, and op are abbreviations for coat protein, activation domain, and operator, respectively. (B) Forty-one clones were sequenced and the aptamers were classified as described in Fig. 2A.

FIGURE 4.

Three-hybrid assay with representative members from all classes. (A) HIS3 expression was monitored as growth on selection plates (SC −his+5 mM 3-AT). Strain L40-coat expressed either Rrm4^AD-G (top) or IRP1^AD-G (bottom). The respective hybrid RNA is indicated next to the various plate sectors. (B) β-galactosidase expression was quantified using a fluorogenic substrate. The difference in relative fluorescence units (RFUs) per minute is given for strains expressing the different proteins (inlay) and hybrid RNAs (bottom) tested.

FIGURE 5.

Identifying the aptamer interaction domain of Rrm4 in vivo. (A) Schematic representation of the Rrm4 domain architecture (top; RRM and C-terminal PABC domains are given as rectangles and oval, respectively) and the introduced point mutations (bottom). The boxed amino acids in the RNA contact region RNP1 were mutated to alanine. The numbers indicate the distance between RNP1 and RNP2. (B) HIS3 expression was monitored as growth on selection plates (SC −his+5 mM 3-AT). Strain L40-coat expressed different versions of Rrm4^AD-G (Rrm4^AD-G-mR1, Rrm4^AD-G-mR2, and Rrm4^AD-G-mR3 carry point mutations in RRM1, RRM2, and RRM3, respectively; labeled with mR1, mR2, and mR3 on the left). The respective hybrid RNA is indicated next to the various plate sectors (labeled on the right). (C) Expression of Rrm4^AD-G versions (given at the bottom) was determined in strains expressing different hybrid RNAs (inlay) by quantifying Gfp fluorescence per OD₆₀₀. (D) Relative β-galactosidase activity was determined using a fluorogenic substrate. To account for differences in protein expression, enzyme activity is given relative to Gfp fluorescence (ΔRFU^Gal per minute per RFU^Gfp). Different versions of Rrm4^AD-G (bottom) were tested with various hybrid RNAs (inlay).