Nucleic acid strand displacement - from DNA nanotechnology to translational regulation

Abstract

Nucleic acid strand displacement reactions involve the competition of two or more DNA or RNA strands of similar sequence for binding to a complementary strand, and facilitate the isothermal replacement of an incumbent strand by an invader. The process can be biased by augmenting the duplex comprising the incumbent with a single-stranded extension, which can act as a toehold for a complementary invader. The toehold gives the invader a thermodynamic advantage over the incumbent, and can be programmed as a unique label to activate a specific strand displacement process. Toehold-mediated strand displacement processes have been extensively utilized for the operation of DNA-based molecular machines and devices as well as for the design of DNA-based chemical reaction networks. More recently, principles developed initially in the context of DNA nanotechnology have been applied for the de novo design of gene regulatory switches that can operate inside living cells. The article specifically focuses on the design of RNA-based translational regulators termed toehold switches. Toehold switches utilize toehold-mediated strand invasion to either activate or repress translation of an mRNA in response to the binding of a trigger RNA molecule. The basic operation principles of toehold switches will be discussed as well as their applications in sensing and biocomputing. Finally, strategies for their optimization will be described as well as challenges for their operation in vivo.

Keywords: RNA synthetic biology; Strand displacement; riboregulators.

Figure 1.

Nucleic acid strand displacement. a) Three-way branch migration occurs, when two identical sequences (green strands, example sequences are given for clarity) compete for binding to a complement. Shown is one of many states explored during branch migration. b) a single branch migration step consists of dissociation of one of the base-pairs at the branch point, followed by reformation of the base-pair with the alternative nucleotide on the competing strand. The process will proceed as an unbiased random walk. In the scheme, N is the total length of the strands and [n,m] denotes a state in which the strands form n and m base-pairs, respectively. c) in toehold-mediated strand displacement (TMSD), the invader contains an additional sequence complementary to the “toehold” on the incumbent-complement complex. This allows the invader to initiate a branch migration process more efficiently, ultimately resulting in displacement of the incumbent strand. d) a similar situation – relevant also to applications in riboregulation - arises when an invader attaches to and invades a nucleic acid secondary structure. Opening of the hairpin loop can be used to activate a sequence domain (blue) for binding.

Figure 2.

Biological processes involving strand displacement. a) Homologous recombination occurring after a double strand break (DSB). The broken strands (green) are trimmed at their 5’ ends with an exonuclease which allows invasion of the intact homologous duplex shown in black. Template-directed DNA polymerization is accompanied by a branch migration process (the newly synthesized sequence is shown in yellow, 4WJ = four-way junction). b) Recombination protein RecA forms a nucleoprotein filament with single-stranded DNA. RecA helps to invade a duplex with a homologous sequence, resulting in the formation of a D-loop. c) a Cas9-guide RNA complex binds to a PAM sequence on a target duplex, from where a strand invasion process is initiated. The RNA spacer sequence (green) displaces one of the DNA strands of the duplex and thereby forms an R-loop [4,5]. .

Figure 3.

Translational toehold switches. a) in a toehold switch, the ribosome binding site (RBS) and translation start codon (AUG) are sequestered in a stable secondary structure and are therefore not accessible for a ribosome. Addition of a trigger RNA results in toehold-mediated strand invasion and opening of the toehold hairpin, which in turn activates translation of the downstream coding region. The hairpin at the 5’ end of the trigger is a measure against RNA degradation [21]. b) Toehold switches can also be created based on the architecture of translational riboswitches. Here the toehold hairpin sequesters an anti-anti-RBS, while the RBS is initially bound to an anti-RBS. Trigger RNA can induce refolding of the structure, which releases the RBS and thus activates translation [41]. c) a translational off switch can be constructed by sequestering the RBS with the help of a trigger. In this case, the hairpin containing the RBS loop is designed to be weak enough to be invaded by the ribosome (opening of the hairpin is indicated by the gray arrows). Trigger RNA locks the hairpin with the RBS and thus switches translation off [42]. d) Eukaryotic mRnas are capped at the 5’ end and do not have an equivalent of the ribosome binding site. Shown is an example of a eukaryotic toehold switch that is based on the accessibility of an internal ribosome entry site (IRES) on an mRNA, which was derived from viral RNA [43]. .

Figure 4.

Applications in sensing and biocomputing. a) Toehold switches have been used in various biosensing applications. For nucleic acid sensing, the analyte contained in the initial sample typically has to be amplified first, the toehold switch then serves as a sequence-specific sensor for the amplicons. The toehold switch can be operated in a cell-free expression system, which can be utilized for low cost sensors [45,46]. b) Several toehold switches can be combined in series, in parallel, or in more complex configurations. Shown is an or gate based on two switches in series. In the presence of trigger A, the first RBS becomes available and the ribosome then opens the second hairpin [47]. c) a trigger for a toehold switch can be split into two parts (trigger a and B), resulting in an and gate configuration. As TMSD requires both a sequence complementary to the toehold and to the sequence to be invaded, translation is only activated in the presence of both triggers [47]. .