Advancing Genetic Alphabet Expansion: Synthesis of 7-(2-Thienyl)-Imidazo[4,5-b]pyridine (Ds) and 4-(4-Pentyne-1,2-diol)-1-Propynyl-2-Nitropyrrole (Diol-Px) for Use in Replicable Unnatural Base Pairs for PCR Applications.

Expanding the genetic alphabet enhances DNA recombinant technologies by introducing unnatural base pairs (UBPs) beyond the standard A-T and G-C pairs, leading to biomaterials with novel and increased functionalities. Recent developments include UBPs that effectively function as a third base pair in replication, transcription, and/or translation processes. One such UBP, Ds-Px, demonstrates extremely high specificity in replication. Chemically synthesized DNA fragments containing Ds bases are amplified by PCR with the 5'-triphosphates of Ds and Px deoxyribonucleosides (dDsTP and dPxTP). The Ds-Px pair system has applications in enhanced DNA data storage, generation of high-affinity DNA aptamers, and incorporation of functional elements into RNA through transcription. This protocol describes the synthesis of the amidite derivative of Ds (dDs amidite), the triphosphate dDsTP, and the diol-modified dPxTP (Diol-dPxTP) for PCR amplifications involving the Ds-Px pair. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of Ds deoxyribonucleoside (dDs) Basic Protocol 2: Synthesis of dDs amidite Basic Protocol 3: Synthesis of dDs triphosphate (dDsTP) Basic Protocol 4: Synthesis of Pn deoxyribonucleoside (4-iodo-dPn) Basic Protocol 5: Synthesis of acetyl-protected diol-modified Px deoxyribonucleoside (Diol-dPx) Basic Protocol 6: Synthesis of Diol-dPx triphosphate (Diol-dPxTP) Basic Protocol 7: Purification of triphosphates Support Protocol 1: Synthesis of Hoffer's chlorosugar Support Protocol 2: Preparation of 0.5 M pyrophosphate in DMF Support Protocol 3: Preparation of 2 M TEAB buffer.

Investigating the Effect of RNA Scaffolds on the Multicolor Fluorogenic Aptamer Pepper in Different Bacterial Species.

RNA synthetic biology tools have primarily been applied in E. coli; however, many other bacteria are of industrial and clinical significance. Thus, the multicolor fluorogenic aptamer Pepper was evaluated in both Gram-positive and Gram-negative bacteria. Suitable HBC-Pepper dye pairs were identified that give blue, green, or red fluorescence signals in the E. coli, Bacillus subtilis, and Salmonella enterica serovar Typhimurium (S. Typhimurium). Furthermore, we found that different RNA scaffolds have a drastic effect on in vivo fluorescence, which did not correlate with the in vitro folding efficiency. One such scaffold termed DF30-tRNA displays 199-fold greater fluorescence than the Pepper aptamer alone and permits simultaneous dual color imaging in live cells.

An electrochemiluminescence microsensor based on DNA-silver nanoclusters amplification for detecting cellular adenosine triphosphate.

Adenosine triphosphate (ATP), as the primary energy source, plays vital roles in many cellular events. Developing an efficient assay is crucial to rapidly evaluate the level of cellular ATP. A portable and integrated electrochemiluminescence (ECL) microsensor array based on a closed bipolar electrode (BPE) was presented. In the BPE unit, the ECL chemicals and oxidation/reduction were separated from the sensing chamber. The ATP aptamer was assembled with single-stranded DNA (ssDNA) in the sensing chamber. ATP capture made the aptamer disassemble from the ssDNA and facilitated DNA-templated silver nanocluster (Ag NC) generation by the target-rolling circle amplification (RCA) reaction. The guanine-rich padlock sequence produced tandem periodic cytosine-rich sequences by the RCA, inducing Ag NC generation in the cytosine-rich region of the produced DNA strands through Ag+ reduction. The in situ Ag NC generation enhanced the circuit conductivity of the BPE and promoted the ECL reaction of [Ru(bpy)2dppz]2+/tripropylamine in the anodic reservoir. On this ECL microsensor, a good linear relationship of ATP was achieved ranging from 30 to 1000 nM. The ATP content in HepG2 cells was selectively and sensitively determined without complex pretreatment. The ATP amount of 25 cells could be successfully detected when a sub-microliter sample was loaded.

Aptamer-mediated double strand displacement amplification with microchip electrophoresis for ultrasensitive detection of Salmonella typhimurium.

Rapid and quantitative detection of foodborne bacteria is of great significance to public health. In this work, an aptamer-mediated double strand displacement amplification (SDA) strategy was first explored to couple with microchip electrophoresis (MCE) for rapid and ultrasensitive detection of Salmonella typhimurium (S. Typhimurium). In double-SDA, a bacteria-identified probe consisting of the aptamer (Apt) and trigger sequence (Tr) was ingeniously designed. The aptamer showed high affinity to the S. Typhimurium, releasing the Tr sequence from the probe. The released Tr hybridized with template C1 chain, initiating the first SDA to produce numerous output strands (OS). The second SDA process was induced with the hybridization of the liberated OS and template C2 sequence, generating a large number of reporter strands (RS), which were separated and quantified through MCE. Cascade signal amplification and rapid separation of nucleic acids could be realized by the proposed double-SDA method with MCE, achieving the limit of detection for S. typhimurium down to 6 CFU/mL under the optimal conditions. Based on the elaborate design of the probes, the double-SDA assisted MCE strategy achieved better amplification performance, showing high separation efficiency and simple operation, which has satisfactory expectation for bacterial disease diagnosis.

Flipping the script: Understanding riboswitches from an alternative perspective.

Riboswitches are broadly distributed regulatory elements most frequently found in the 5'-leader sequence of bacterial mRNAs that regulate gene expression in response to the binding of a small molecule effector. The occupancy status of the ligand-binding aptamer domain manipulates downstream information in the message that instructs the expression machinery. Currently, there are over 55 validated riboswitch classes, where each class is defined based on the identity of the ligand it binds and/or sequence and structure conservation patterns within the aptamer domain. This classification reflects an "aptamer-centric" perspective that dominates our understanding of riboswitches. In this review, we propose a conceptual framework that groups riboswitches based on the mechanism by which RNA manipulates information directly instructing the expression machinery. This scheme does not replace the established aptamer domain-based classification of riboswitches but rather serves to facilitate hypothesis-driven investigation of riboswitch regulatory mechanisms. Based on current bioinformatic, structural, and biochemical studies of a broad spectrum of riboswitches, we propose three major mechanistic groups: (1) "direct occlusion", (2) "interdomain docking", and (3) "strand exchange". We discuss the defining features of each group, present representative examples of riboswitches from each group, and illustrate how these RNAs couple small molecule binding to gene regulation. While mechanistic studies of the occlusion and docking groups have yielded compelling models for how these riboswitches function, much less is known about strand exchange processes. To conclude, we outline the limitations of our mechanism-based conceptual framework and discuss how critical information within riboswitch expression platforms can inform gene regulation.

Selection of internalizing RNA aptamers into human breast cancer cells derived from primary sites.

Breast cancer is the most common cancer in women. Although chemotherapy is still broadly used in its treatment, adverse effects remain a challenge. In this scenario, aptamers emerge as a promising alternative for theranostic applications. Studies using breast cancer cell lines provide useful information in laboratory and preclinical investigations, most of which use cell lines established from metastatic sites. However, these cell lines correspond to cell populations of the late stage of tumor progression. On the other hand, studies using breast cancer cells established from primary sites make it possible to search for new theranostic approaches in the early stages of the disease. Therefore, this work aimed to select RNA aptamers internalized by MGSO-3 cells, a human breast cancer cell line, derived from a primary site previously established in our laboratory. Using the Cell-Internalization SELEX method, we have selected two candidate aptamers (ApBC1 and ApBC2). We evaluated their internalization efficiencies, specificities, cellular localization by Reverse Transcription-qPCR (RT-qPCR) and confocal microscopy assays. The results suggest that both aptamers were efficiently internalized by human breast cancer cells, MACL-1, MDA-MB-231, and especially by MGSO-3 cells. Furthermore, both aptamers could effectively distinguish human breast cancer cells derived from normal human mammary cell (MCF 10A) and prostate cancer cell (PC3) lines. Therefore, ApBC1 and ApBC2 could be promising candidate molecules for theranostic applications, even in the early stages of tumor progression.

Fluorogenic RNA Aptamer-Based Amplification and Transcription Strategy for Label-free Sensing of Methyltransferase Activity in Complex Matrixes.

DNA methyltransferase is significant in cellular activities and gene expression, and its aberrant expression is closely linked to various cancers during initiation and progression. Currently, there is a great demand for reliable and label-free techniques for DNA methyltransferase evaluation in tumor diagnosis and cancer therapy. Herein, a low-background fluorescent RNA aptamer-based sensing approach for label-free quantification of cytosine-guanine (CpG) dinucleotides methyltransferase (M.SssI) is reported. The fluorogenic light-up RNA aptamers-based strategy exhibits high selectivity via restriction endonuclease, padlock-based recognition, and RNA transcription. By combining rolling circle amplification (RCA), and RNA transcription with fluorescence response of RNA aptamers of Spinach-dye compound, the proposed platform exhibited efficiently ultrahigh sensitivity toward M.SssI. Eventually, the detection can be achieved in a linear range of 0.02-100 U mL-1 with a detection limit of 1.6 × 10-3 U mL-1. Owing to these superior features, the method is further applied in serum samples spiked M.SssI, which delivers a recovery ranging from 92.0 to 107.0% and a relative standard deviation <7.0%, providing a promising and practical tool for determining M.SssI in complex biological matrices.

High-affinity truncated aptamers for detection of Cronobacter spp with magnetic separation-assisted DNAzyme-driven 3D DNA walker.

After optimizing the original aptamer sequence by truncation strategy, a magnetic separation-assisted DNAzyme-driven 3D DNA walker fluorescent aptasensor was developed for detecting the food-borne pathogen Cronobacter species. Iron oxide magnetic nanoparticles (MNPs) modified with a hybrid of truncated aptamer probe and DNAzyme strand (AP-E1) denoted as MNPs@AP-E1, were employed as capture probes. Simultaneously, a DNAzyme-driven 3D-DNA walker was utilized as the signal amplification element. The substrate strand (Sub) was conjugated with the gold nanoparticles (AuNPs), resulting in the formation of AuNPs@Sub, which served as a 3D walking track. In the presence of the target bacteria and Mg2+, E1-DNAzyme was activated and moved along AuNPs@Sub, continuously releasing the signal probe. Under optimized conditions, a strong linear correlation was observed for Cronobacter sakazakii (C. sakazakii) in the concentration range 101 to 106 CFU mL-1, with a low detection limit of 2 CFU mL-1. The fluorescence signal responses for different Cronobacter species exhibited insignificant differences, with a relative standard deviation of 3.6%. Moreover, the aptasensor was successfully applied to determine  C. sakazakii in real samples with recoveries of 92.86%-108.33%. Therefore, the novel method could be a good candidate for ultra-sensitive and selective detection of Cronobacter species without complex manipulation.

Highly sensitive and specific graphene oxide-based FRET aptasensor for quantitative detection of human soluble growth stimulating gene protein 2.

Soluble growth stimulation expressed gene 2 (sST2) is a new generation biomarker in the diagnosis and prognosis of heart failure (HF). Here, the sST2-specific aptamers were selected from a random ssDNA library with the full length of 88 nucleotides (nt) via target-immobilized magnetic beads (MB)-based systematic evolution of ligands by exponential enrichment (SELEX) technology. After eight rounds of selection, six aptamers with the most enrichment were selected. Among, the aptamer L1 showed the high-affinity binding to sST2 with the lowest Kd value (77.3 ± 0.05 nM), which was chosen as the optimal aptamer for further molecular docking. Then, the aptamer L1 was used to construct a graphene oxide (GO) - based fluorescence resonance energy transfer (FRET) biosensor for sST2, which exhibits a linear detection range of 0.1-100 µg/ml and a detection limit of 3.7 ng/ml. The aptasensor was applied to detect sST2 in real samples, with a good correlation and agreement with the traditional enzyme-linked immunosorbent assay (ELISA) when quantitative analyzing the sST2 concentration in serum samples from HF patients. The results show that not only an efficient strategy for screening the practicable aptamer, but also a rapid and sensitive detection platform for sST2 were established.

Fluorescent aptasensor for highly sensitive detection of Staphylococcus aureus based on dual-amplification strategy by integrating DNA walking and hybridization chain reaction.

Food-borne diseases caused by bacteria threaten human health. Herein, we presented a new fluorescent aptasensor by coupling DNA walking and hybridization chain reaction (HCR) for convenient and sensitive quantification of bacteria. Staphylococcus aureus (S. aureus) was selected as target. When there was target in the system, the binding of S. aureus with its aptamer caused the disintegration of aptamer/DNA walker on the surface of AuNPs and released DNA walker. With the help of Nt.BsmAI, DNA walker moved along the surface of AuNPs and trigger probe was detached from AuNPs. The trigger probe could initiate hybridization chain reaction (HCR) and opened the stems of H1@AuNPs probe and H2@AuNPs probe. After the addition of nicking endonuclease, the adjacent upconversion nanoparticles (UCNPs, NaYF4:Yb3+, Er3+) were further away from the quenchers (AuNPs) of H1 and H2. Therefore, the fluorescence intensity of UCNPs could be restored via fluorescence resonance energy transfer (FRET). Bacteria were thus detected by recording the fluorescence intensity of UCNPs. This method is simple, rapid and sensitive. It can directly detect bacteria in a low background signal. The limit of detection (LOD) was 10 CFU/mL, detection time was less than 3 h. Recovery rates in simulated milk, honey and human serum samples ranged from 93.6 % to 105.8 %. The strategy opens up new paths for early diagnosis of diseases and food monitoring.

Split G-quadruplex based PfAgo sensing platform for nucleotide mutation discrimination and human genotyping.

A PfAgo-G4 sensing platform exploiting G4 as a signal reporter was proposed, validated, and optimized. By introducing two mismatches at the Link strand, a universal nucleotide design rule was established for accurate single nucleotide polymorphism discrimination with PfAgo-G4. The FUT2 gene was then successfully and accurately genotyped using human buccal swab samples.

Label-free and highly sensitive detection of microRNA from cancer cells via target-induced cascade amplification generation of lighting-up RNA aptamers.

The abnormal expression levels of miRNAs have been proven to be highly related to the generation of various diseases and are also closely associated with the stages and types of disease development. The novel RNA aptamers-based homogenous fluorescent methods were simple, with low background signal and high signal-to-noise ratio, but lacked effective signal amplification technology to achieve sensitive detection of trace miRNA markers. There is an urgent need for combining effective nucleic acid amplification technology with RNA aptamer to achieve highly sensitive and accurate detection of miRNA. For this purpose, a new DNA multi-arm nanostructure-based dual rolling circle transcription machinery for the generation of lighting-up MG RNA aptamers is constructed for label-free and highly sensitive sensing of miRNA-21. In this system, the target miRNA-21 induces a structural transformation of the DNA multi-arm nanostructure probe to recycle miRNA-21 and trigger two independent rolling circle transcription reactions to generate two long RNAs, which can partially hybridize with each other to generate large amounts of complete MG RNA aptamers. These RNA aptamers can associate with organic MG dye to produce significantly enhanced fluorescence signals to accomplish ultrasensitive miRNA-21 detection down to 0.9 fM. In addition, this method exhibits high selectivity to distinguish miRNA-21 even with single nucleotide mismatch, and also has potential application capability to monitor different expression levels of miRNA-21 from different cancer cells. The effective collaboration between MG RNA aptamer and rolling circle transcription reaction makes this fluorescent method show the significant advantages of low background signal, high signal-to-noise ratio and high detection sensitivity. It has great potential to be a promising means to achieve label-free and highly sensitive monitoring of other trace biological markers via a simple change of target sequence.

Reversible Aptamer Staining, Sorting, and Cleaning of Cells (Clean FACS) with Antidote Oligonucleotide or Nuclease Yields Fully Responsive Cells.

The ability to reverse the binding of aptamers to their target proteins has received considerable attention for developing controllable therapeutic agents. Recently, use of aptamers as reversible cell-sorting ligands has also sparked interest. Antibodies are currently utilized for isolating cells expressing a particular cell surface receptor. The inability to remove antibodies from isolated cells following sorting greatly limits their utility for many applications. Previously, we described how a particular aptamer-antidote oligonucleotide pair can isolate cells and clean them. Here, we demonstrate that this approach is generalizable; aptamers can simultaneously recognize more than one cell type during fluorescent activated cell sorting (FACS). Moreover, we describe a novel approach to reverse aptamer binding following cell sorting using a nuclease. This alternative strategy represents a cleaning approach that does not require the generation of antidote oligonucleotides for each aptamer and will greatly reduce the cost and expand the utility of Clean FACS.

Optimization of RNA Pepper Sensors for the Detection of Arbitrary RNA Targets.

The development of fluorescent light-up RNA aptamers (FLAPs) has paved the way for the creation of sensors to track RNA in live cells. A major challenge with FLAP sensors is their brightness and limited signal-to-background ratio both in vivo and in vitro. To address this, we develop sensors using the Pepper aptamer, which exhibits superior brightness and photostability when compared to other FLAPs. The sensors are designed to fold into a low fluorescence conformation and to switch to a high fluorescence conformation through toehold or loop-mediated interactions with their RNA target. Our sensors detect RNA targets as short as 20 nucleotides in length with a wide dynamic range over 300-fold in vitro, and we describe strategies for optimizing the sensor's performance for any given RNA target. To demonstrate the versatility of our design approach, we generated Pepper sensors for a range of specific, biologically relevant RNA sequences. Our design and optimization strategies are portable to other FLAPs and offer a promising foundation for future development of RNA sensors with high specificity and sensitivity for detecting RNA biomarkers with multiple applications.

Nucleic acid aptamer based thermally oxidized porous silicon/zinc oxide microarray chip for detection of ochratoxin A in cereals.

A nucleic acid aptamer based thermally oxidized porous silicon/zinc oxide microarray chip was constructed for the detection of ochratoxin A. The hybrid chains formed by aptamer and complementary chains labeled with fluorescent groups and fluorescent burst groups were used as recognition molecules, and the detection of toxins was accomplished on the chip by the principle of fluorescence signal burst and recovery. The modified QuEChERS method was used for sample pretreatment and the performance of the method was evaluated. The results showed that the linear range was 0.02 âˆ¼ 200 ng/kg with the detection limit of 0.0196 ng/kg under the optimal detection conditions. The method was applied to different cereals with the recoveries of 90.30 âˆ¼ 111.69 %. The developed microarray chip has the advantages of being cost-effective, easy to prepare, sensitive and specific, and can provide a new method for the detection of other toxins.

Simultaneous detection of AFB1 and aflD gene by "Y" shaped aptamer fluorescent biosensor based on double quantum dots.

The developed method for simultaneous detection of aflatoxin B1 (AFB1) and aflD genes can effectively monitor from the source and reduce the safety problems and economic losses caused by the production of aflatoxin, which can be of great significance for food safety regulations. In this paper, we constructed a sensitive and convenient fluorescent biosensor to detect AFB1 and aflD genes simultaneously based on fluorescence resonance energy transfer (FRET) between quantum dots (QDs) and a black hole quenching agent. A stable "Y" shaped aptasensor was employed as the detection platform and a double quantum dot labeled DNA fragment was utilized to be the sensing element in this work. When the targets of AFB1 and aflD genes were presented in the solution, the aptamer in the "Y" shaped probe is specifically recognized by the target. At this time, both Si-carbon quantum dots (Si-CDs) and CdTe QDs are far away from the BHQ1 and BHQ3 to recover the fluorescence. The linear range of the prepared fluorescence simultaneous detection method was as wide as 0.5-500 ng·mL-1 with detection lines of 0.64 ng·mL-1 for AFB1 and 0.5-500 nM with detection lines of 0.75 nM for aflD genes (3σ/k). This fabricated fluorescent biosensor was further validated in real rice flour and corn flour samples, which also achieved good results. The recoveries were calculated by comparing the known and found amounts of AFB1 which ranged from 88.4 to approximately 115.32% in the rice flour samples and 90.7 ~ 102.58% in the corn flour samples. The recoveries of aflD genes ranged from 84.32 to approximately 109.3% in the rice flour samples and 89.48 ~ 100.99% in the corn flour samples. Therefore, the proposed biosensor can significantly improve food safety and quality control through a simple, fast, and sensitive agricultural product monitoring and detection system.

G-triplex/hemin DNAzyme mediated colorimetric aptasensor for Escherichia coli O157:H7 detection based on exonuclease III-assisted amplification and aptamers-functionalized magnetic beads.

Escherichia coli O157: H7 (E. coli O157: H7) is one of the most common foodborne pathogens and is widespread in food and the environment. Thus, it is significant for rapidly detecting E. coli O157: H7. In this study, a colorimetric aptasensor based on aptamer-functionalized magnetic beads, exonuclease III (Exo III), and G-triplex/hemin was proposed for the detection of E. coli O157: H7. The functional hairpin HP was designed in the system, which includes two parts of a stem containing the G-triplex sequence and a tail complementary to cDNA. E. coli O157: H7 competed to bind the aptamer (Apt) in the Apt-cDNA complex to obtain cDNA. The cDNA then bound to the tail of HP to trigger Exo III digestion and release the single-stranded DNA containing the G-triplex sequence. G-triplex/hemin DNAzyme could catalyze TMB to produce visible color changes and detectable absorbance signals in the presence of H2O2. Based on the optimal conditions, E. coli O157: H7 could be detected down to 1.3 × 103 CFU/mL, with a wide linear range from 1.3 × 103 to 1.3 × 107 CFU/mL. This method had a distinguished ability to non-target bacteria, which showed good specificity. In addition, the system was successfully applied to detect E. coli O157: H7 in milk samples.

RNA aptamers developed against tRip: A preliminary approach targeting tRNA entry in Plasmodium.

Malaria is caused by Plasmodium parasites that multiply inside host cells and can be lethal when P. falciparum is involved. We identified tRip as a membrane protein that facilitates the import of exogenous transfer RNA (tRNA) into the parasite. tRip encompasses a tRNA binding domain exposed on the parasite surface. We used the SELEX approach to isolate high-affinity and specific tRip-binding RNA motifs from a library of random 25 nucleotide-long sequences. In five rounds of combined negative and positive selections, an enriched pool of aptamers was obtained; sequencing revealed that they were all different in their primary sequence; only by comparing their structure predictions did most of the selected aptamers reveal a conserved 5-nucleotide motif sequence. We showed that the integral motif is essential for tRip-binding while the rest of the molecule can be significantly reduced or mutated as long as the motif is presented in a single-stranded region. Such RNA aptamers bind in place of the original tRNA substrate and act as an efficient competitor, suggesting that they can block tRip function and slow parasite development.

Aptamer-modified tetrahedral DNA nanostructure-immobilized liposome for specific gene delivery and potential cancer theragnostic.

Targeted delivery of therapeutic agents to cancer cells is crucial for effective cancer treatment without adverse effects. In this study, we developed a novel delivery carrier, Aptamer-modified tetrahedral DNA nanostructure (TDN) immobilized Liposome (ApTL), for specific delivery to nucleolin-overexpressing cancer cells. We demonstrated that targeted ApTL was highly effective in delivering plasmid and mRNA to nucleolin-overexpressing cancer cells compared to non-targeted ApTL with a non-specific aptamer. ApTL, which is highly negative and nano-sized, specifically delivered nucleic acids to MDA-MB-231 and HeLa cancer cells, primarily via lipid-raft-mediated endocytosis. Furthermore, the co-delivery of mRNA and doxorubicin resulted in increased apoptosis and reduced cancer cell viability. Interestingly, co-delivery of mRNA and Dox did not show a significant difference in EGFP expression at 24 h but dramatically increased EGFP expression at 48 h, making ApTL/mEGFP/Dox a promising candidate for detecting live cancer cells after targeted cancer drug treatment. Our results suggest that ApTL can be a promising tool for the targeted delivery of therapeutic agents to nucleolin-overexpressing cancer cells, providing a new strategy for cancer theragnostic.

RNA Aptamer-Mediated Gene Activation Systems for Inducible Transgene Expression in Animal Cells.

RNA expression analyses can be used to obtain various information from inside cells, such as physical conditions, the chemical environment, and endogenous signals. For detecting RNA, the system regulating intracellular gene expression has the potential for monitoring RNA expression levels in real time within living cells. Synthetic biology provides powerful tools for detecting and analyzing RNA inside cells. Here, we devised an RNA aptamer-mediated gene activation system, RAMGA, to induce RNA-triggered gene expression activation by employing an inducible complex formation strategy grounded in synthetic biology. This methodology connects DNA-binding domains and transactivators through target RNA using RNA-binding domains, including phage coat proteins. MS2 bacteriophage coat protein fused with a transcriptional activator and PP7 bacteriophage coat protein fused with the tetracycline repressor (tetR) can be bridged by target RNA encoding MS2 and PP7 stem-loops, resulting in transcriptional activation. We generated recombinant CHO cells containing an inducible GFP expression module governed by a minimal promoter with a tetR-responsive element. Cells carrying the trigger RNA exhibited robust reporter gene expression, whereas cells lacking it exhibited no expression. GFP expression was upregulated over 200-fold compared with that in cells without a target RNA expression vector. Moreover, this system can detect the expression of mRNA tagged with aptamer tags and modulate reporter gene expression based on the target mRNA level without affecting the expression of the original mRNA-encoding gene. The RNA-triggered gene expression systems developed in this study have potential as a new platform for establishing gene circuits, evaluating endogenous gene expression, and developing novel RNA detectors.