Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers

Abstract

Nucleic acid circuits are finding increasing real-life applications in diagnostics and synthetic biology. Although DNA has been the main operator in most nucleic acid circuits, transcriptionally produced RNA circuits could provide powerful alternatives for reagent production and their use in cells. Towards these goals, we have implemented a particular nucleic acid circuit, catalytic hairpin assembly, using RNA for both information storage and processing. Our results demonstrated that the design principles developed for DNA circuits could be readily translated to engineering RNA circuits that operated with similar kinetics and sensitivities of detection. Not only could purified RNA hairpins perform amplification reactions but RNA hairpins transcribed in vitro also mediated amplification, even without purification. Moreover, we could read the results of the non-enzymatic amplification reactions using a fluorescent RNA aptamer 'Spinach' that was engineered to undergo sequence-specific conformational changes. These advances were applied to the end-point and real-time detection of the isothermal strand displacement amplification reaction that produces single-stranded DNAs as part of its amplification cycle. We were also able to readily engineer gate structures with RNA similar to those that have previously formed the basis of DNA circuit computations. Taken together, these results validate an entirely new chemistry for the implementation of nucleic acid circuits.

Figure 1.

Design of non-enzymatic catalyzed RNA hairpin assembly circuit. (a) Schematic of catalyzed nucleic acid hairpin assembly circuit adapted from (2). The circuit composed of hairpins H1 and H2 is turned on in the presence of the input sequence (C1). C1 catalyzes the assembly of H1 and H2 into an H1:H2 duplex and is itself recycled. Circuit output (H1:H2 duplex) is quantitated as increasing fluorescence intensity of a labeled oligonucleotide probe (RepF) on displacement of its complementary quencher oligonucleotide (RepQ) by the H1:H2 duplex. (b) Design of T7 RNA polymerase-driven transcription templates for enzymatic synthesis of RNA CHA circuit components with precise 5′- and 3′-ends. Transcription template for each component, H1, H2 and C1, is flanked on both the left (L) and the right (R) sides by hammerhead ribozymes (HRz). The size (in nucleotides) of each component and its ribozyme flanks is indicated under each schematic. Secondary structures of the resulting chimeric RNA at 42°C before ribozyme processing are depicted (green, A; blue, C; black, G; red, U). The RNA structures were generated using NUPACK (13–16).

Figure 2.

Synthesis and execution of RNA CHA circuit. (a) LHRz and RHRz-mediated cotranscriptional RNA cleavage releases the internal circuit components H1, H2 and C1. Fifity nanograms of PCR-generated transcription templates for H1, H2 and C1 was transcribed in 50 µl of reactions by T7 RNA polymerase for 2 h at 42°C. Two microliters of the resulting transcripts was analyzed by electrophoresis on a 10% denaturing polyacrylamide gel. Single-stranded DNA oligonucleotides were used as size markers. (b) RNA hairpins undergo catalyzed assembly into RNA duplexes. Gel-purified RNA catalyst C1 and the hairpins H1 and H2 were combined as indicated and incubated in 1× TNaK buffer containing 20 U of RNaseOUT for 150 min at 42°C (lanes 1–4), 52°C (lanes 5–8) or 62°C (lanes 9–12). The reactions were then analyzed on a 10% native polyacrylamide gel. Fifteen nanograms of C1 RNA was included in lane 13 as a control. Single-stranded DNA oligonucleotides were used as size markers.

Figure 3.

Kinetics and sensitivity of purified RNA CHA circuit. (a) Fold amplification and sensitivity of gel-purified RNA CHA circuit. The RNA CHA circuit can detect pure C1 to picomolar concentration with ∼87-fold amplification of 0.1 nM C1 within 315 min at 52°C. Circuit output measured as concentration of RepF released from RepF:RepQ duplex was extrapolated from a standard curve of free RepF. (b) Initial rate of C1-catalyzed H1:H2 hybridization was measured by incubating varying concentrations of gel-purified H1 and H2 with 2.5 nM pure C1 RNA diluted in 1 µM oligo dT₁₇. Circuits were executed in 1× TNaK buffer containing 20 U of RNaseOUT, 0.5 µM ROX reference dye and 400 nM RepF annealed with 5× excess (2 µM) RepQ at 52°C for 315 min. Initial rates were calculated from circuit output measurements made during the initial 3–20 min of circuit operation. Average data from three separate experiments are represented. H1 concentration has a greater impact on the initial rate suggesting that the first step of the circuit (C1-triggered unfolding of H1) is a rate limiting step. (c) Effect of H1 and H2 concentrations on the kinetics of RNA CHA circuit. Average raw fluorescence data from triplicate experiments are plotted. Circuit output is maximal when operated with near equal concentrations of H1 and H2. Increasing H2 concentration above that of H1 generally decreased the initial reaction rate and resulted in reduced circuit output.

Figure 4.

Cotranscriptional RNA CHA and circuit design optimization for cotranscription. (a) Cotranscribed RNA circuit components undergo catalyzed hairpin assembly without requiring gel purification of individual reactants. Fifty nanograms each of H1 and H2 transcription templates, along with titrating amounts of C1 transcription template, was cotranscribed for 1 h at 42°C using T7 RNA polymerase followed by passage through Illustra MicroSpin Sephadex G25 columns. Transcription templates were amplified from cloned inserts using primers pCR2.1.F and pCR2.1.R specific to plasmid sequences flanking the inserts. Two microliter aliquots of the cotranscribed RNA mixtures were then incubated in 15 µl of volume with 400 nM RepF annealed with 5× excess (2 µM) RepQ fluorescent DNA reporter duplex in 1× TNaK buffer containing 20 U of RNaseOUT and 0.5 µM ROX reference dye to quantitate formation of H1:H2 RNA duplexes at 52°C. Average data from triplicate experiments are represented. (b and c) Schematic depicting sequences of RNA hairpins H1 and H2 with one- or two-base engineered mismatches. Mismatched H1 (mH1) presents a two-base mismatch between its domain 4* and domain 4 of H2. The hairpins mAH1 and mGH1 each contain a single mismatched base between their domain 4* and the domain 4 of H2. The mutated H2 hairpin m2H2 presents two mismatched bases between its domain 2* and the H1 domain 2.

Figure 5.

Operation of cotranscriptionally generated RNA CHA circuits without any downstream purification and design optimization for detection of DNA target. (a) Fifty nanograms each of the indicated pairs of hairpin 1 and 2 transcription templates was cotranscribed with or without 10 ng of C1 transcription template for 1 h at 42°C using T7 RNA polymerase. Following transcription, 2 µl of the reaction mix was directly incubated in 1× TNaK buffer containing 20 U of RNaseOUT and 0.5 µM ROX reference dye along with 400 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time at 52°C. (b) Schematic depicting SDA of DNA. The single-stranded template DNA (black arrow) consists of a sequence (C*) complementary to the RNA CHA catalyst followed by the nicking enzyme recognition sequence (NE) that is present on the non-cleaved DNA strand and a primer binding site. Following primer binding (step 1), the DNA polymerase synthesizes the complementary strand that now completes the duplex NE site and contains the RNA CHA catalyst sequence (C). Nicking enzyme then binds the duplex NE site (step 2) and cleaves the newly synthesized strand at the NE site. The new 3′-OH group generated at the nick site is then extended by the DNA polymerase (step 3) while displacing the previously synthesized strand. The displaced ssDNA amplicon can then catalyze RNA CHA. (c) Schematic of DNA target sequence design for catalysis of RNA CHA. Single toehold (domain 1*) DNA target C1 (generated by SDA from the template TLTRSDA) with the same domain architecture as the RNA C1 is an inefficient catalyst of RNA CHA. Extended DNA target C1234 (generated by SDA from the template 1234LTRSDA) presenting two toeholds for RNA H1 successfully catalyzes RNA CHA.

Figure 6.

Cotranscriptionally generated RNA CHA as signal transducer for nucleic acid diagnostics. (a) End-point sequence-specific detection of SDA-generated ssDNA targets by RNA CHA. Samples with or without 10 nM template 1234LTRSDA were amplified by SDA for 90 min at 37°C in 25 µl of reaction volumes. Reactions were then incubated at 95°C for 5 min and stored at room temperature before assay by RNA CHA. Five microliters of these SDA products was then probed with 2 µl of Sephadex G25 column-purified cotranscribed mH1:H2 RNA CHA circuit. RNA CHA cotranscriptions were performed with T7 RNA polymerase using 50 ng each of the mH1 and H2 transcription templates for 1 h at 42°C. End-point RNA CHA detection reactions were assembled in 1× TNaK buffer containing 20 U of RNaseOUT, 0.5 µM ROX reference dye and 100 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time at 52°C. Negative control reactions lacking RNA CHA components or containing 2 µl of either only mH1 or H2 were also tested. (b) Real-time signal transduction of ssDNA-generating SDA by cotranscribed mH1:H2 RNA CHA. High temperature (55°C) SDA reactions were set up with or without 10 nM 1234HTRSDA template in 20 µl of volume containing 0.5 µM ROX reference dye and 75 nM RepF (annealed with 5× excess RepQ) fluorescent DNA reporter duplex for quantitating RNA CHA in real-time. Real-time sequence-specific signal transduction was achieved by adding 2 µl of unpurified mH1:H2 RNA CHA circuits cotranscribed from 50 ng of each transcription template to the SDA reactions. Control SDA reactions containing no RNA CHA components or 2 µl of either only mH1 or H2 were also tested.

Figure 7.

Reengineering of the fluorophore-dependent fluorescent RNA aptamer Spinach into sequence-triggered aptamer beacons (Spinach.ST). The fluorophore DFHBI bound to Spinach.ST1 is indicated as a red stellate. Spinach.ST is embedded within a transfer RNA scaffold and is therefore not subjected to RNA end processing by hammerhead ribozymes.

Figure 8.

An entirely RNA-based CHA circuit operation and fluorimetric detection. (a) CHA circuit components (hairpins H1B and H2 and catalyst C1) and the RNA reporter Spinach.ST1 were separately transcribed by T7 RNA polymerase from 500 ng of PCR-generated duplex DNA transcription templates. H1B, H2 and C1 transcription templates were amplified using primers complementary to the exact ends of the cloned inserts (H1B.amp.F:H1B.amp.R, H2.amp.F:H2.amp.R and C1.amp.F:C1.amp.R, respectively) rather than the flanking plasmid. Spinach.ST transcription templates were amplified using primers specific to the flanking plasmid sequence at the 5′-end (pCR2.1.F) and the primer sphT.U.R specific to the 3′-end sequence of Spinach.ST. Transcription reactions were filtered through Sephadex G25 columns before circuit assembly. Three microliters of H1B, H2, C1 and Spinach.ST1 transcripts was mixed in indicated combinations and incubated in 1× TNaK buffer containing 70 µM DFHBI and 20 U of RNaseOUT. Circuit output was measured as increasing fluorescence intensity over time at 37°C. (b–d) Performance of DNA reporter duplex H1BF:H1BQ (b) versus Spinach.ST1 (c) in measuring RNA CHA circuit output. Indicated concentrations of gel-purified RNA hairpins H1B and H2 were incubated with equal concentration of H1BF:H1BQ or gel-purified Spinach.ST1 (+ 70 µM DFHBI) in the presence of titrating concentrations of pure C1 RNA. All circuits were operated in 1× TNaK buffer containing 20 U of RNaseOUT at 37°C, and average data from triplicate experiments are represented. Signal-to-noise ratio of H1BF:H1BQ versus Spinach.ST1 over the time course of RNA CHA detection is plotted in (d).

Figure 9.

Application of RNA CHA circuit as an OR logic processor. (a) Schematic of RNA CHA circuit operation in response to either catalyst C1 OR C2. The RNA hairpin H1B serves as the OR gate, and circuit output is measured fluorimetrically using Spinach.ST1 RNA aptamer beacon. (b) Circuit components (H1B and H2 RNA hairpins), reporter RNA (Spinach.ST1) and the inputs C1 and C2 were transcribed from 500 ng of duplex DNA transcription templates using T7 RNA polymerase. Transcription templates were prepared using the same procedure as Figure 8. Following filtration through Sephadex G25, 3 µl/transcript (or 1.5 µl each of C1 and C2 when added together in a reaction) was mixed in the indicated combinations in 1× TNaK buffer containing 70 µM DFHBI and 20 U of RNaseOUT. Circuits were operated at 37°C, and outputs were measured fluorimetrically.