T7 RNA Polymerase Research Articles

Effective Synthesis of High-Integrity mRNA Using In Vitro Transcription

: mRNA vaccines are entering a period of rapid development. However, their synthesis is still plagued by challenges related to mRNA impurities and fragments (incomplete mRNA). Most impurities of mRNA products transcribed in vitro are mRNA fragments. Only full-length mRNA transcripts containing both a 5′-cap and a 3′-poly(A) structure are viable for in vivo expression. Therefore, RNA fragments are the primary product-related impurities that significantly hinder mRNA efficacy and must be effectively controlled; these species are believed to originate from either mRNA hydrolysis or premature transcriptional termination. In the manufacturing of commercial mRNA vaccines, T7 RNA polymerase-catalyzed in vitro transcription (IVT) synthesis is a well-established method for synthesizing long RNA transcripts. This study identified a pivotal domain on the T7 RNA polymerase that is associated with erroneous mRNA release. By leveraging the advantageous properties of a T7 RNA polymerase mutant and precisely optimized IVT process parameters, we successfully achieved an mRNA integrity exceeding 91%, thereby further unlocking the immense potential of mRNA therapeutics.

Functionalizing DNA Origami by Triplex-Directed Site-Specific Photo-Cross-Linking.

Here, we present a cross-linking approach to covalently functionalize and stabilize DNA origami structures in a one-pot reaction. Our strategy involves adding nucleotide sequences to adjacent staple strands, so that, upon assembly of the origami structure, the extensions form short hairpin duplexes targetable by psoralen-labeled triplex-forming oligonucleotides bearing other functional groups (pso-TFOs). Subsequent irradiation with UVA light generates psoralen adducts with one or both hairpin staples leading to site-specific attachment of the pso-TFO (and attached group) to the origami with ca. 80% efficiency. Bis-adduct formation between strands in proximal hairpins further tethers the TFO to the structure and generates "superstaples" that improve the structural integrity of the functionalized complex. We show that directing cross-linking to regions outside of the origami core dramatically reduces sensitivity of the structures to thermal denaturation and disassembly by T7 RNA polymerase. We also show that the underlying duplex regions of the origami core are digested by DNase I and thus remain accessible to read-out by DNA-binding proteins. Our strategy is scalable and cost-effective, as it works with existing DNA origami structures, does not require scaffold redesign, and can be achieved with just one psoralen-modified oligonucleotide.

Mechanistic modeling of in vitro transcription incorporating effects of magnesium pyrophosphate crystallization.

The in vitro transcription (IVT) reaction used in the production of messenger RNA vaccines and therapies remains poorly quantitatively understood. Mechanistic modeling of IVT could inform reaction design, scale-up, and control. In this work, we develop a mechanistic model of IVT to include nucleation and growth of magnesium pyrophosphate crystals and subsequent agglomeration of crystals and DNA. To help generalize this model to different constructs, a novel quantitative description is included for the rate of transcription as a function of target sequence length, DNA concentration, and T7 RNA polymerase concentration. The model explains previously unexplained trends in IVT data and quantitatively predicts the effect of adding the pyrophosphatase enzyme to the reaction system. The model is validated on additional literature data showing an ability to predict transcription rates as a function of RNA sequence length.

Synthesis and Biological Profiling of Quinolino-Fused 7-Deazapurine Nucleosides.

A series of quinolino-fused 7-deazapurine (pyrimido[5',4':4,5]pyrrolo[3,2-f]quinoline) ribonucleosides were designed and synthesized. The synthesis of the key 11-chloro-pyrimido[5',4':4,5]pyrrolo[3,2-f]quinoline was based on the Negishi cross-coupling of iodoquinoline with zincated 4,6-dichloropyrimidine followed by azidation and thermal or photochemical cyclization. Vorbrüggen glycosylation of the tetracyclic heterocycle followed by cross-coupling or substitution reactions at position 11 gave the desired set of final nucleosides that showed moderate to weak cytostatic activity and fluorescent properties. The corresponding fused adenosine derivative was converted to the triphosphate and successfully incorporated to RNA using in vitro transcription with T7 RNA polymerase.

Comprehensive evaluation of T7 promoter for enhanced yield and quality in mRNA production

The manufacturing of mRNA vaccines relies on cell-free based systems that are easily scalable and flexible compared with the traditional vaccine manufacturing processes. Typically, standard processes yield 2 to 5 g L−1 of mRNA, with recent process optimisations increasing yields to 12 g L−1. However, increasing yields can lead to an increase in the production of unwanted by-products, namely dsRNA. It is therefore imperative to reduce dsRNA to residual levels in order to avoid intensive purification steps, enabling cost-effective manufacturing processes. In this work, we exploit sequence modifications downstream of the T7 RNA polymerase promoter to increase mRNA yields whilst simultaneously minimising dsRNA. In particular, transcription performance was optimised by modifying the sequence downstream of the T7 promoter with additional AT-rich sequences. We have identified variants that were able to produce higher amounts of mRNA (up to 14 g L−1) in 45 min of reaction. These variants exhibited up to a 30% reduction in dsRNA byproduct levels compared to a wildtype T7 promoter, and have similar EGFP protein expression. The results show that optimising the non-coding regions can have an impact on mRNA production yields and quality, reducing overall manufacturing costs.

Functional predictability of universal gene circuits in diverse microbial hosts

AbstractAlthough the principles of synthetic biology were initially established in model bacteria, microbial producers, extremophiles and gut microbes have now emerged as valuable prokaryotic chassis for biological engineering. Extending the host range in which designed circuits can function reliably and predictably presents a major challenge for the concept of synthetic biology to materialize. In this work, we systematically characterized the cross‐species universality of two transcriptional regulatory modules—the T7 RNA polymerase activator module and the repressors module—in three non‐model microbes. We found striking linear relationships in circuit activities among different organisms for both modules. Parametrized model fitting revealed host non‐specific parameters defining the universality of both modules. Lastly, a genetic NOT gate and a band‐pass filter circuit were constructed from these modules and tested in non‐model organisms. Combined models employing host non‐specific parameters were successful in quantitatively predicting circuit behaviors, underscoring the potential of universal biological parts and predictive modeling in synthetic bioengineering.

A sensitive fluorescence biosensor based on ligation-transcription and CRISPR/Cas13a-assisted cascade amplification strategies to detect the H1N1 virus.

We propose a sensitive H1N1 virus fluorescence biosensor based on ligation-transcription and CRISPR/Cas13a-assisted cascade amplification strategies. Products are generated via the hybridization of single-stranded DNA (ssDNA) probes containing T7 promoter and crRNA templates to a target RNA sequence using SplintR ligase. This generates large crRNA quantities in the presence of T7 RNA polymerase. At such crRNA quantities, ternary Cas13a, crRNA, and activator complexes are successfully constructed and activate Cas13a to enhance fluorescence signal outputs. The biosensor sensitively and specifically monitored H1N1 viral RNA levels down to 3.23 pM and showed good linearity when H1N1 RNA concentrations were 100 pM-1 µM. Biosensor specificity was also excellent. Importantly, our biosensor may be used to detect other viral RNAs by altering the sequences of the two probe junctions, with potential applications for the clinical diagnosis of viruses and other biomedical studies.

A Prebiotic Genetic Nucleotide as an Early Darwinian Ancestor for Pre-RNA Evolution.

Prebiotic genetic nucleotides (PGNs) often outcompete canonical alphabets in the formation of nucleotides and subsequent RNA oligomerization under early Earth conditions. This indicates that the early genetic code might have been dominated by pre-RNA that contained PGNs for information transfer and catalysis. Despite this, deciphering pre-RNAs' capacity to acquire function and delineating their evolutionary transition to a canonical RNA World has remained under-researched in the origins of life (OoL) field. We report the synthesis of a prebiotically relevant nucleotide (BaTP) containing the noncanonical nucleobase barbituric acid. We demonstrate the first instance of its enzymatic incorporation into an RNA, using a T7 RNA polymerase. BaTP's incorporation into baby spinach aptamer allowed it to retain its overall secondary structure and function. Finally, we also demonstrate faithful transfer of information from the pre-RNA-containing BaTP to DNA, using a high-fidelity RNA-dependent DNA polymerase, alluding to how selection pressures and complexities could have ensued during the molecular evolution of the early genetic code.

Effective synthesis of circRNA via a thermostable T7 RNA polymerase variant as the catalyst.

Circular RNAs (circRNAs) are endogenous noncoding RNAs (ncRNAs) with transcriptional lengths ranging from hundreds to thousands. circRNAs have attracted attention owing to their stable structure and ability to treat complicated diseases. Our objective was to create a one-step reaction for circRNA synthesis using wild-type T7 RNA polymerase as the catalyst. However, T7 RNA polymerase is thermally unstable, and we streamlined circRNA synthesis via consensus and folding free energy calculations for hotspot selection. Because of the thermal instability, the permuted intron and exon (PIE) method for circRNA synthesis is conducted via tandem catalysis with a transcription reaction at a low temperature and linear RNA precursor cyclization at a high temperature. To streamline the process, a multisite mutant T7 RNA polymerase (S430P, N433T, S633P, F849I, F880Y, and G788A) with significantly improved thermostability was constructed, and G788A was used. The resulting mutant exhibited stable activity at 45°C for over an hour, enabling the implementation of a one-pot transcription and cyclization reaction. The simplified circRNA production process demonstrated an efficiency comparable to that of the conventional two-step reaction, with a cyclization rate exceeding 95% and reduced production of immunostimulatory dsRNA byproducts.

Development of an improved reverse genetics system for avian metapneumovirus (aMPV): A novel vaccine vector protects against aMPV and infectious bursal disease virus

Avian metapneumovirus (aMPV), a paramyxovirus, causes acute respiratory diseases in turkeys and swollen head syndrome in chickens. This study established a reverse genetics system for aMPV subtype B LN16-A strain based on T7 RNA polymerase. Full-length cDNA of the LN16-A strain was constructed by assembling five cDNA fragments between the T7 promoter and hepatitis delta virus ribozyme. Transfection of this plasmid, along with the supporting plasmids encoding the N, P, M2-1, and L proteins of LN16-A into BSR-T7/5 cells, resulted in the recovery of aMPV subtype B. To identify an effective insertion site, the enhanced green fluorescent protein (EGFP) gene was inserted into different sites of the LN16-A genome to generate recombinant LN16-As. The results showed that the expression levels of EGFP at the site between the G and L genes of LN16-A were significantly higher than those at the other two sites (between the leader and N genes or replacing the SH gene). To verify the availability of the site between G and L for foreign gene expression, the VP2 gene of very virulent infectious bursal disease virus (vvIBDV) was inserted into this site, and recombinant LN16-A (rLN16A-vvVP2) was successfully rescued. Single immunization of specific-pathogen-free chickens with rLN16A-vvVP2 induced high levels of neutralizing antibodies and provided 100% protection against the virulent aMPV subtype B and vvIBDV. Establishing a reverse genetics system here provides an important foundation for understanding aMPV pathogenesis and developing novel vector vaccines.

A novel electrochemical biosensor for B-type natriuretic peptide detection based on CRISPR/Cas13a and chain substitution reaction

B-type natriuretic peptide (BNP) is a biomarker for heart failure, a serious and prevalent disease that requires rapid and accurate diagnosis. In this study, we developed a novel electrochemical biosensor for BNP detection based on CRISPR/Cas13a and chain substitution reaction. The biosensor consists of a DNA aptamer that specifically binds to BNP, a T7 RNA polymerase that amplifies the signal, a CRISPR/Cas13a system that cleaves the target RNA, and a two-dimensional DNA nanoprobe that generates an electrochemical signal. The biosensor exhibits high sensitivity, specificity, and stability, with a detection limit of 0.74 aM. The biosensor can also detect BNP in human serum samples with negligible interference, demonstrating its potential for clinical and point-of-care applications. This study presents a novel strategy for integrating CRISPR/Cas13a and chain substitution reaction into biosensor design, offering a versatile and effective platform for biomolecule detection.

Promoting the production of challenging proteins via induced expression in CHO cells and modified cell-free lysates harboring T7 RNA polymerase and mutant eIF2α

Chinese hamster ovary (CHO) cells are crucial in biopharmaceutical production due to their scalability and capacity for human-like post-translational modifications. However, toxic proteins and membrane proteins are often difficult-to-express in living cells. Alternatively, cell-free protein synthesis can be employed. This study explores innovative strategies for enhancing the production of challenging proteins through the modification of CHO cells by investigating both, cell-based and cell-free approaches. A major result in our study involves the integration of a mutant eIF2 translation initiation factor and T7 RNA polymerase into CHO cell lysates for cell-free protein synthesis. This resulted in elevated yields, while eliminating the necessity for exogenous additions during cell-free production, thereby substantially enhancing efficiency. Additionally, we explore the potential of the Rosa26 genomic site for the integration of T7 RNA polymerase and cell-based tetracycline-controlled protein expression. These findings provide promising advancements in bioproduction technologies, offering flexibility to switch between cell-free and cell-based protein production as needed.

One-pot, one-step, label-free miRNA detection method based on the structural transition of dumbbell probe

In this study, we present a one-pot, one-step, label-free miRNA detection method through a structural transition of a specially designed dumbbell-shape probe, initiating a rolling circle transition (RCT). In principle, target miRNA binds to right loop of the dumbbell probe (DP), which allows structural change of the DP to circular form, exposing a sequence complementary to the T7 promoter (T7p) previously hidden within the stem. This exposure allows T7 RNA polymerase to initiate RCT, producing a repetitive Mango aptamer sequence. TO1-biotin, fluorescent dye, binds to the aptamer, inducing a detectable enhancement of fluorescence intensity. Without miR-141, the DP stays closed, RCT is prevented, and the fluorescence intensity remains low. By employing this novel strategy, target miRNA was successfully identified with a detection of 73 pM and a dynamic linear range of 0–10 nM. Additionally, the method developed enables one-pot, one-step, and label-free detection of miRNA, demonstrating potential for point-of-care testing (POCT) applications. Furthermore, the practical application of the designed technique was demonstrated by reliably detecting the target miRNA in the human serum sample. We also believe that the conceived approach could be widely used to detect not only miRNAs but also diverse biomolecules by simply replacing the detection probe.

ASIA: An automated stress-inducible adaptor for enhanced stress protein expression in engineered Escherichia coli.

Orthogonal T7 RNA polymerase (T7RNAP) and T7 promoter is a potent technique for protein expression in broad cells, but the energy requirements associated with this method impede the growth, leading to cell lysis when dealing with toxic and stress proteins. A Lemo21(DE3) strain denoted as L21 offers a solution by fine-tuning T7RNAP levels under rhamnose to induce T7 lysozyme (LysY) and enhance the protein production, but it requires optimization of inducer concentration, cultural temperature, and condition, even the types of carbon sources. Herein, we construct an automated stress-inducible adaptor (ASIA) employing different stress-inducible promoters from Escherichia coli. The ASIA system is designed to automatically regulate LysY expression in response to stress signals, thereby suppressing T7RNAP and amplifying the overexpression of stress protein cutinase ICCM. This approach fine-tunes T7RNAP levels and outperforms L21 in various temperatures and carbon source conditions. The ASIAhtp strain maintains ICCM yield at 91.6 mg/g-DCW even in the limiting carbon source at 1 g/L, which is 12-fold higher in protein productivity compared to using L21. ASIA as a versatile and robust tool for enhancing overexpression of stress proteins in E. coli is expected to address more difficult proteins in the future.

Misacylation of tRNA with Ser-Pro Dipeptide for In Vitro Transcription-Translation.

Serine-proline (Ser-Pro) backbone-modified dipeptide analogues are powerful tools to investigate the role of cis-trans isomerization in the regulation of the cell cycle and transcription. These studies have previously been limited to synthetic peptides, whose synthesis is a challenge for larger peptides due to the compounding yield loss incurred in each step. We now introduce a method for the aminoacylation of tRNA with dipeptides and dipeptide analogs to permit the installation of cis- and trans-locked Ser-Pro analogues into full-length proteins. To that end, we synthesized the 3,5-dinitrobenzyl (DNB)-activated esters of a native Ser-Pro dipeptide and its cis- and trans-locked alkene analogs. Murakami etal. created the DNB flexizyme (dFx), a ribozyme that acylates tRNA with DNB esters of amino acids to permit unnatural amino acids to be incorporated into proteins. A tRNA from yeast that recognizes the amber stop codon, along with the dFx flexizyme, were generated by in vitro transcription with T7 RNA polymerase. dFx was used to successfully catalyze the chemical misacylation of truncated amber tRNA with the Ser-Pro-DNB activated dipeptide. This method allows the introduction of non-native Ser-Pro dipeptide mimics into full-length proteins by in vitro transcription-translation. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 3,5-dinitrobenzyl activated esters of Ser-Pro Basic Protocol 2: Preparation of truncated amber tRNA Basic Protocol 3: Acylation of amber-tRNA by the dFx flexizyme Basic Protocol 4: PAGE electrophoresis of tRNASerPro.

Rational design of oligonucleotides for enhanced in vitro transcription of small RNA.

All kinds of RNA molecules can be produced by in vitro transcription using T7 RNA polymerase using DNA templates obtained by solid-phase chemical synthesis, primer extension, PCR or DNA cloning. The oligonucleotide design, however, is a challenge to non-experts as this relies on a set of rules that have been established empirically over time. Here, we describe a Python program to facilitate the Rational design of Oligonucleotides, Calculated with Kinetic parameters for Enhanced in vitro Transcription (ROCKET). The Python tool employs thermodynamic parameters, performs folding-energy calculations, and selects oligonucleotides suitable for the polymerase extension reaction. These oligonucleotides improve yields of template DNA. With the oligonucleotides selected by the program, the tRNA transcripts can be prepared by a one-pot reaction of the DNA polymerase extension reaction and the transcription reaction. Also, the ROCKET-selected oligonucleotides provide greater transcription yields than that from oligonucleotides selected by Primerize, a leading software for designing oligonucleotides for in vitro transcription, due to the enhancement of template DNA synthesis. Apart from over 50 tRNA genes tested, an in vitro transcribed self-cleaving ribozyme was found to have catalytic activity. In addition, the program can be applied to the synthesis of mRNA, demonstrating the wide applicability of the ROCKET software.

Polymerase and Proofreading Exonuclease Domains in the DNA Polymerase γ and Nuclear-encoded RNA Polymerase of the Human Mitochondria and Identification of Mitochondrial Disease Mutations in the Polymerase Active Site Region of DNA Polymerase γ

Recent reports have made it clear that mutations in the mitochondrial DNA (mtDNA) polymerase γ (POLG1) are a major cause of many human diseases. Mutations in the mtDNA polymerase leads to defective oxidative phosphorylation and ATP production, resulting in many mitochondrial diseases. For their mitochondrial genome replication, humans and animals use POLG1, whose catalytic site is essentially similar to the E. coli DNA polymerase I (DNA pol I). Multiple sequence alignment (MSA) analysis have shown that the POLG1 and E. coli DNA pol I use identical amino acids in their polymerase catalytic sites, viz. –943R-4EHAKI1FNYGRI955Y8G- (human DNA pol γ) as –R-4RSAKA1INFGLIY8G- (E. coli DNA pol I). However, the human POLG1 shows only 31.25% identity with the E. coli DNA pol I, suggesting a highly divergent evolution. Mutation(s) in the POLG1 gene is one of the most common causes of many inherited mitochondrial diseases in children and adults. Depending on their location within the enzyme, mutations either lead to mtDNA depletion or accumulation of multiple mtDNA deletions leading to various mitochondrial diseases. The most common POLG1 dominant mutation, viz. Y955→H/C, which lead to a severe, early-onset of multi-systemic mitochondrial disease with bilateral sensorineural hearing loss, cataract, myopathy, and liver failure is located in the template-binding pair of the polymerase catalytic site region by MSA. Another dominant mutation, R943→H/C is observed in patients with Progressive External Ophthalmoplegia (adPEO, an autosomal, dominant, heritable mitochondrial disorder) is located in the nucleoside triphosphate (NTP) selection amino acid in the polymerase catalytic site region. The nuclear-encoded RNA polymerase (NEP) is imported from the nucleus and involves in the transcription of all the mitochondrial genes. The human mitochondrial NEP showed 39.30%, 40.12% and 26.98% identities to the NEPs of the mitochondria and chloroplasts of Arabidopsis thaliana, and T7 RNA polymerase, respectively, suggesting that the human and plant mitochondrial NEPs are distinctly different. Interestingly, the human NEP’s catalytic core is almost completely conserved in the plant mitochondrial and chloroplast NEPs, viz. –R-4KVVKQ1TVMTVVY8G- (Human) and –R-4KLVKQ1TVMTSVY8G- (A. Thaliana). Furthermore, the mitochondrial NEP’s catalytic core from human and different animal sources is remarkably conserved and is in close agreement with other NEPs of plant sources. Both the human DNA pol γ and NEP possess a typical template-binding pair (-YG-), a basic catalytic amino acid (K) to initiate catalysis and a basic nucleotide selection amino acid R at -4 from the catalytic K. The PR exonucleases of POLG1 and NEP belong to the DEDD-superfamily of exonucleases and uses a Y or H as the proton acceptor, respectively.

Urea supplementation improves mRNA in vitro transcription by decreasing both shorter and longer RNA byproducts

ABSTRACT The current letter to the editor describes the presence of RNA byproducts in small-scale in vitro transcription (IVT) reactions as evaluated by capillary gel electrophoresis, asymmetric flow field flow fractionation, immunoblotting, cell-free translation assays, and in IFN reporter cells. We compare standard T7 RNA polymerase (RNAP) based IVT reactions to two recently described protocols employing either urea supplementation or using the VSW3 RNAP. Our results indicate that urea supplementation yields considerably less RNA byproducts and positively affects the overall number of full-length transcripts. In contrast, VSW3 IVT reactions demonstrated a low yield and generated a higher fraction of truncated transcripts. Lastly, both urea mRNA and VSW3 mRNA elicited considerably less IFN responses after transfection in mouse macrophages.

Exploring T7 RNA polymerase-assisted CRISPR/Cas13a amplification for the detection of BNP via electrochemiluminescence sensing platform

Brain natriuretic peptide (BNP) is considered to be an important biomarker of heart failure (HF) attracting attention. However, its low concentration and short half-life in blood lead to a low-sensitivity detection of BNP, which is a challenge that has to be overcome. In this work, we propose a highly specific, highly sensitive T7 RNA polymerase-assisted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a system to detect BNP via an electrochemiluminescence (ECL) sensing platform and incorporate exonuclease III (Exo III)-hairpin and dumbbell-shaped hybridization chain reaction (HCR) technologies. In this detection scheme, the ECL sensing platform possesses low background signal and high sensitivity. Firstly, the T7 promoter-initiated T7 RNA polymerase acts as a signal amplification technique to generate large amounts of RNAs that can activate CRISPR/Cas13a activity. Secondly, CRISPR/Cas13a is able to trans-cleave the surrounding trigger strand to produce DNA1. Thirdly, DNA1 is involved in the co-amplification reaction of Exo III and hairpin DNA, which subsequently triggers a dumbbell-shaped HCR technology. Eventually, a large number of Ru (II) molecules are inserted into the interstitial space of the dumbbell-shaped HCR to generate a strong ECL signal. The CRISPR/Cas13a possesses outstanding specificity for a single base and increased sensitivity. The tightly conformed dumbbell-shaped HCR provides higher sensitivity than the traditional linear HCR amplification technique. Ultimately, the clever combination of several amplification reactions enables the limit of detection (LOD) as low as 3.2 fg/mL. It showed promise for clinical sample testing, with recovery rates ranging from 98.4% to 103% in 5% human serum samples. This detection method offered a valuable tool for early HF detection, emphasizing the synergy of amplification strategies and specificity conferred by CRISPR/Cas13a technology.

Synthesis and Enzymatic Incorporation of a Dual-App Nucleotide Probe That Reports Antibiotics-Induced Conformational Change in the Bacterial Ribosomal Decoding Site RNA.

Natural nucleosides are nonfluorescent and do not have intrinsic labels that can be readily utilized for analyzing nucleic acid structure and recognition. In this regard, researchers typically use the so-called "one-label, one-technique" approach to study nucleic acids. However, we envisioned that a responsive dual-app nucleoside system that harnesses the power of two complementing biophysical techniques namely, fluorescence and 19F NMR, will allow the investigation of nucleic acid conformations more comprehensively than before. We recently introduced a nucleoside analogue by tagging trifluoromethyl-benzofuran at the C5 position of 2'-deoxyuridine, which serves as an excellent fluorescent and 19F NMR probe to study G-quadruplex and i-motif structures. Taking forward, here, we report the development of a ribonucleotide version of the dual-app probe to monitor antibiotics-induced conformational changes in RNA. The ribonucleotide analog is derived by conjugating trifluoromethyl-benzofuran at the C5 position of uridine (TFBF-UTP). The analog is efficiently incorporated by T7 RNA polymerase to produce functionalized RNA transcripts. Detailed photophysical and 19F NMR of the nucleoside and nucleotide incorporated into RNA oligonucleotides revealed that the analog is structurally minimally invasive and can be used for probing RNA conformations by fluorescence and 19F NMR techniques. Using the probe, we monitored and estimated aminoglycoside antibiotics binding to the bacterial ribosomal decoding site RNA (A-site, a very important RNA target). While 2-aminopurine, a famous fluorescent nucleic acid probe, fails to detect structurally similar aminoglycoside antibiotics binding to the A-site, our probe reports the binding of different aminoglycosides to the A-site. Taken together, our results demonstrate that TFBF-UTP is a very useful addition to the nucleic acid analysis toolbox and could be used to devise discovery platforms to identify new RNA binders of therapeutic potential.