Multistrand Structure Prediction of Nucleic Acid Assemblies and Design of RNA Switches

Abstract

RNA is an attractive material for the creation of molecular logic gates that release programmed functionalities only in the presence of specific molecular interaction partners. Here we present HyperFold, a multistrand RNA/DNA structure prediction approach for predicting nucleic acid complexes that can contain pseudoknots. We show that HyperFold also performs competitively compared to other published folding algorithms. We performed a large variety of RNA/DNA hybrid reassociation experiments for different concentrations, DNA toehold lengths, and G+C content and find that the observed tendencies for reassociation correspond well to computational predictions. Importantly, we apply this method to the design and experimental verification of a two-stranded RNA molecular switch that upon binding to a single-stranded RNA toehold disease-marker trigger mRNA changes its conformation releasing an shRNA-like Dicer substrate structure. To demonstrate the concept, connective tissue growth factor (CTGF) mRNA and enhanced green fluorescent protein (eGFP) mRNA were chosen as trigger and target sequences, respectively. In vitro experiments confirm the formation of an RNA switch and demonstrate that the functional unit is being released when the trigger RNA interacts with the switch toehold. The designed RNA switch is shown to be functional in MDA-MB-231 breast cancer cells. Several other switches were also designed and tested. We conclude that this approach has considerable potential because, in principle, it allows the release of an siRNA designed against a gene that differs from the gene that is utilized as a biomarker for a disease state.

Keywords: Dicer; RNA interference; RNA switch; RNA/DNA hybrid; secondary structure.

Figure 1.

Schematic (a) and 2D (b) representation of the interaction between the functional antitarget strand (red), an antitrigger strand (blue) and a trigger strand (yellow). The antitarget strand and the antitrigger strand form a duplex that represents a nonfunctional unit. The trigger mRNA strand (yellow) interacts with the RNA toehold (blue); this reaction leads to the formation of a duplex between the antitrigger strand and the trigger strand (“waste”) as well as a reformed antitarget strand that represents a functional Dicer substrate RNA (“functional unit”).

Figure 2.

Schematic representation of the secondary structure search space (a) and search algorithm (b). The example shown in (a) corresponds to a hypothetical secondary structure that consists of two fully extended helices. The folding status of each helix is represented by one integer number that can be 0 (helix is unfolded), 1 (helix is fully base paired), 2 or 3 (helix is halfway folded from one end). The completely unfolded secondary structure is for two hypothetical helices represented as “0,0” (first row), while the structure “1,1” (see red arrow) corresponds to a secondary structure with both helices fully base paired. This leads for the case of two helices to 16 possible structural alternatives. The search algorithm depicted in (b) consists of two priority queues were the lowest-free energy structures have the highest priority for exploring additional folding events. During one stage structures are removed from search queue 1 (the “emitting queue”), and multiple structures with additional helices are generated and placed into search queue 2 (the “receiving queue”). This process is continued until search queue 1 is empty, after which search queue 2 becomes the emitting queue and search queue 1 becomes the receiving queue. This process is continued until no more helices can be placed. The search queues are “leaky” in the sense that when a maximum number of containing structures is exceeded, the structures with the least favorable free energy are discarded. During the search process, the free energies of the found structures are analyzed in order to compute concentrations and in order to identify the structure with the lowest free energy.

Figure 3.

Schematic representation of the secondary structure model (a) and three-dimensional model (b) of the duplex between the antitarget strand (red) and the antitrigger strand (blue). A predicted interaction between the toehold region of the antitrigger and the 3′ end of the antitarget strand (shown in Figure S3) is not part of the 3D model.

Figure 4.

Box-whisker plots of the prediction quality (Matthews Correlation Coefficients) of HyperFold, pknotsRG, and UnaFold corresponding to a set of 110 pseudoknotted RNAs (top) and 741 nonpseudoknotted RNAs (bottom) obtained from the RNA Strand Database. The box plots group data corresponding to RNA lengths of 1−50, 51−100, 101−150, 151−200, and 201−250 nt.

Figure 5.

Scatter plot of experimentally measured fraction of reassociating RNA/DNA hybrid complexes (“Reassociation”) as a function of computationally predicted free energy of toehold binding. The size, color, and shape of the plotted symbols depict the toehold length, G+C content, and strand concentration, respectively. The plotted relative amounts of reassociation are means over experimental data values (for the same strand concentration and toehold length) as depicted also in Figure S2. The reason for the choice of predicted free energy of toehold binding as coordinate axis is that the initial duplex formation of cognate toeholds acts as transition state. As expected, the higher the free-energy barrier of the transition state (right side of diagram), the lower the reaction rate and (for a given fixed time point) the lower the relative amount of the reaction products (y-axis). An analysis of the kinetics of RNA-toehold formation has been performed in a related publication (Afonin et al. The use of minimal RNA toeholds to trigger the activation of multiple functionalities. Accepted to this Journal).

Figure 6.

Experimental verification of RNA switch formation and function by total RNA staining native-PAGE experiments. (a) Results demonstrate formation of a two-stranded switch and its further interaction with the mRNA fragment leading to the release of DS RNA containing shRNA-like structure (refolded antitarget strand). (b) Results for Dicer experiments carried out for switch and released antitarget strand respectively with recombinant Dicer using the human turbo Dicer enzyme kit (Genlantis). (c and d) Results show that the communication between the switch and mRNA is initiated through the toehold interaction. As negative controls, switches without a toehold (c) or with a mutated toehold (d) were tested. Red boxes indicate the bands corresponding to the released Dicer substrate RNA.

Figure 7.

RNA switch mediated gene silencing in human breast cancer cells. Enhanced green fluorescent protein (eGFP) knockdown assays were performed using MDA-MB-231 cells that stably express eGFP. Cells were transfected with the CTGF-eGFP RNA switch, or the individual antitarget strand or antitrigger strand that compose the switch. All transfections were performed at 50 nM [RNA]. Cells transfected with Lipofectamine 2000 only (no RNA) served as a negative control. Levels of eGFP expression were assessed 3 days post-transfection by (a) flow cytometry and (b) fluorescence microscopy. (c) Mean and standard error for flow cytometry fluorescence experiments for cells transfected with 0 nM and 5 nM CTGF siRNA 2 days prior to transfection with the RNA switch. Black: eGFP fluorescence for switch (50 nM); Red: eGFP fluorescence for antitarget strand (50 nM). One can see that the complete switch leads to a higher response in eGFP expression as a function of CTGF trigger knockdown.