In this paper, a method to discriminate between two target RNA sequences that differ by one nucleotide only is presented. The method relies on the formation of alternative structures, i.e., quadruplex–duplex hybrid (QDH) and duplex with dangling ends (Dss), after hybridization of DNA or RNA G-rich oligonucleotides with target sequences containing 5′–GGGCUGG–3′ or 5′–GGGCGGG–3′ fragments. Using biophysical methods, we studied the effect of oligonucleotide types , non-nucleotide modifications , and covalently attached G4 ligand on the ability of G-rich oligonucleotides to assemble a G-quadruplex motif. We demonstrated that all examined non-nucleotide modifications could mimic the external loops in the G-quadruplex domain of QDH structures without affecting their stability. Additionally, some modifications, in particular the presence of two abasic residues in the G-rich oligonucleotide, can induce the formation of non-canonical QDH instead of the Dss structure upon hybridization to a target sequence containing the GGGCUGG motif. Our results offer new insight into the sequential requirements for the formation of G-quadruplexes and provide important data on the effects of non-nucleotide modifications on G-quadruplex formation. G-quadruplexes G4) are structures that have received significant attention in recent years. The canonical, unimolecular G4 structures are formed from DNA or RNA G-quadruplex forming sequences (G4FS) comprising four G-tracts, and they fold into stacks of at least two guanine tetrads stabilized by Hoogsteen hydrogen bonds. Four G-tracts are necessary to form a G-quadruplex; however, they can also be part of two (bimolecular G4) or four separate strands (tetramolecular G4) . The fol are struf G4 RNA ,10,11. Mf G4 RNA ,16,17,18f G4 RNA ,25,26,27f G4 RNA . Environf G4 RNA . It was f G4 RNA . At presf G4 RNA . This G4f G4 RNA . Using af G4 RNA . In addif G4 RNA ,36,37,38f G4 RNA ,33. The f G4 RNA . The lonf G4 RNA . G-quadrf G4 RNA ,45,46,47f G4 RNA ,49. Two f G4 RNA ,52,53,54f G4 RNA ,56. For f G4 RNA .Previous reports suggested that most RNA G-quadruplexes exist in living cells in an unfolded state; however, recent studies proposed a model of a dynamic G4 RNA folding equilibrium controlled mainly by ions and G-quadruplex binding proteins ,57,58,59UGG–3′ vs. 5′–GGGCGGG–3′) can be targeted by G-rich oligonucleotides (QD) in a structure-specific manner. The change in a single nucleotide from uridine to guanosine results in the formation of a second 3 nt GGG tract in a target sequence and triggers the formation of a G-quadruplex motif , which was removed from the solid support and deprotected by overnight treatment with concentrated ammonia and ethanol (3:1) at 55 °C. Next, the solvent was evaporated to dryness, and the 2′-silyl protection was removed by treatment with 1.0 M triethylammonium fluoride at 65 °C for 2.5 h. In the next step, the precipitation was made by the addition of 5 mL of 1-butanol, and the samples were stored at −20 °C for 1 h. The precipitate was separated from the solution by spinning at 5000 rpm, 4 °C, for 10 min. The MMTr group was removed using 80% acetic acid/water for 30 min, and the solvent was evaporated to dryness. The NH2-CCC oligonucleotide was precipitated in the presence of 2% NaClO4/acetone. All oligonucleotides (4HCO3/H2O) and buffer B (100% CH3CN) at a 1.5 mL/min flow rate, 70 °C. The buffer gradient was as follows: (1) 0–2 min 0% B; (2) 2–10 min 0–10% B; (3) 10–12 min 10–50% B; (4) 12–13 min 50–0% B; and (5) 13–30 min 0% B. UV detection was performed at λmax 268 nm. Synthesis of DMT-ON DNA and RNA oligonucleotides at the 1.0 µmole scale was performed according to routine procedures on a MerMade12  or a Gene World DNA synthesizer  under the conditions recommended by the manufacturer. Samples were cleaved from the solid support, deprotected using standard procedures , Sterling, VA, USA). The only exception was MMTr-C6-amino oligoribonucleotide  by loading on the filter, washing several times with 4000 µL MilliQ water, and then washing successively against ∼150 mM LiCl and against water. All oligonucleotides were desalted using AmiconTo prevent dimerization of G-quadruplexes, experiments were performed in the presence of 50 mM KCl instead of 150 mM KCl ,52. 2-CCC according to a published procedure [o-BMVC-C3-NHS was coupled to the NHrocedure . The purFirst, 350 pmole RNA oligo-o-BMVC-C3 was suspended in 4 µL water and 4 µL of 8 M urea . Next, the sample was heated at 95 °C for 4 min, cooled to room temperature, and loaded on 15% TBE-Urea gels . Denaturing gel electrophoresis experiments were performed in 1.5 × TBE buffer. The electrophoresis experiment was run at 180 V for 2 h, and the gel was visualized by UV shadowing .First, 350 pmole of oligonucleotides were suspended in 50 mM KCl, 10 mM phosphate potassium buffer, pH 6.8 or 150 mM NaCl, 10 mM phosphate sodium buffer, pH 6.8 . Next, samples were heated at 95 °C for 3 min and gradually cooled to room temperature. Subsequently, samples were mixed with 2 µL of 50% glycerol and loaded on 20% TBE gels . O’RangeRuler 5 bp and DNA ladder containing 50 and 100 bp as brighter bands  were used as molecular markers. Gel electrophoresis experiments performed in a 0.5 × TBE buffer were run at 160 V for ~2 h at 4 °C (an ice bath), and the gels were first visualized by UV shadow (image) and then stained by N-methyl mesoporphyrin IX . After post-staining the gels with NMM, the gels were scanned with a Fuji FLA-5100 imaging system . T hybrid). The last gel was viewed by UV shadow to visualize all RNA (image) and exposed to 473 nm light to visualize fluorescently labeled G-quadruplex  equipped with a QCI CryoProbe. The oligonucleotides were annealed by heating to 90 °C and then slowly cooled to room temperature. The T and QD/UT oligonucleotides were dissolved in a buffer containing 50 mM KCl, 10 mM phosphate potassium buffer, or 150 mM NaCl, 10 mM phosphate sodium buffer at pH 6.8. Thermal denaturation curves were obtained by monitoring at 260 and 295 nm with a JASCO V-650 spectrophotometer  using quartz optical cuvettes of 0.5 path length with the sample volume of 150 µL. Samples were protected against evaporation by silicone oil. Before measurements, the cuvettes filled with samples were spun at 5000 revolutions per minute for 3 min to avoid the formation of air bubbles during measurements. The temperature range was 20–90 °C, using a scan rate of 0.5 °C min−1. Spectra were processed and prepared using Origin 8 Software . The melting temperature was determined by the local maximum of the first derivatives of the absorbance vs. temperature curve.First, 590 pmole of QD/GT or QD/UT (25 µmol/L) in a buffer containing 10 mM potassium phosphate, 50 mM KCl, 6.8 pH at 25 °C. Fluorescence spectra were measured from 550 to 750 nm with a 2 nm step, 800 V detector sensitivity using 400 nm excitation. Fluorescence spectra were recorded using a JASCO J-815 CD/fluorescence spectropolarimeter .One equivalent of NMM was mixed with QD/G2 mmol−1), without normalization by the number of residues in the oligonucleotide. Spectra were processed and prepared using the Origin 8 Software. CD spectra of oligonucleotides were recorded using a JASCO J-815 spectropolarimeter  equipped with a temperature controller. For each sample, 3 spectral scans were accumulated at 25 °C over wavelengths in the range from 220 to 320 nm. Samples of each oligonucleotide were prepared at a concentration of 8.0 μM in 50 mM KCl, 10 mM potassium phosphate buffer, or 150 mM NaCl, 10 mM sodium phosphate buffer at 6.8 pH using a 0.5 cm path length quartz cuvette with a volume of 1750 µL. Next, samples were prepared by heating the oligonucleotides at 90 °C for 5 min and gradually cooling to room temperature. CD spectra were expressed in the units of molar ellipticity Δɛ , the CGGGCGGGC G-quadruplex (2Q), and the 14 bp duplex (DX). In the presence of K+ ions, the NMR spectrum of CCC/GT displayed imino proton signals in both duplex and G-quadruplex regions, and the peak distribution patterns resembled that of the individual components, duplex, and G-quadruplex. This was consistent with the formation of the quadruplex–duplex hybrid structure (QDH) by CCC/GT. When CCC RNA oligonucleotide was hybridized to the UT target, only signals characteristic of the imino protons involved in the formation of Watson–Crick base pairs were observed. This suggests that the replacement of one of the guanosine residues by uridine, such as the UT target, can prevent G-quadruplex formation  were in agreement with the formation of the hairpin structures as predicted by the RNAstructure software  in the presence of 50 mM KCl to assess the molecularity of CCC/Gomplexes . The bansoftware B. As expo CCC/GT , lane 5′T suggested its parallel topology [T differed from that of CCC/GT only in the intensity of the band at ~265 nm. Unfortunately, CD spectra of parallel G-quadruplexes are very similar to those of A-form duplex structures. As a result, they have little use in structural analysis of RNA quadruplex–duplex hybrids [The binding of NMM to the G-quadruplex domain of CCC/Gtopology ,72, whic hybrids . T and CCC/UT structures were determined by analyzing UV-melting profiles at 260 and 295 nm in the presence of potassium cations  confirmed the G4-dependent stabilization effect.The thermal stabilities of CCC/G cations . The melectively . The proectively . A similT and UT RNA targets in the presence of potassium cations.Next, we compared the tendency of G-rich RNA and DNA molecules to form QDH and Dss structures. We hybridized the DNA equivalent of the CCC sequence, DNA-CCC , with GT1H NMR spectra of two DNA:RNA hetero-complexes: DNA-CCC/UT and DNA-CCC/GT. The spectrum of the DNA-CCC/GT displayed signals both in duplex (11.5–15 ppm) and G-quadruplex (10.5–12 ppm) regions, indicating the formation of a hybrid quadruplex–duplex structure. The 1H NMR spectrum of DNA-CCC/UT showed that signals in the region of 10.5–12 ppm characteristic of G-quadruplex formation were missing, thus suggesting the formation of a Dss structure. However, comparison of the 1H NMR spectrum of DNA-CCC/UT with DNA-CCC and UT clearly showed that it was a sum of signals characteristic of two separate strands, i.e., DNA-CCC and UT, thus suggesting that the structure of a DNA: RNA heteroduplex with dangling ends was not formed , and single-strand DNA-CCC (lane 6) relative to RNA homo-complexes CCC/GT and CCC/UT (lanes 2–3). Bands corresponding to bimolecular structures DNA-CCC/GT and DNA-CCC/UT migrated faster than their RNA:RNA counterparts. The smaller intensity of the band corresponding to DNA-CCC/UT (lane 5) than that of CCC/UT (lane 3), and the presence of unhybridized single-strands DNA-CCC and UT in lane 5, were indicative of a poor hybridization of DNA to RNA strands. When the gel was post-stained with NMM, fluorescence bands were observed only for CCC/GT and DNA-CCC/GT and indicated the formation of the G-quadruplex domain in both cases (T indicated the formation of higher-order structures (HOS)   in the presence of K+, in addition to the imino protons from Watson–Crick base pairs, broad resonances were observed in the 10.5–12.0 ppm region for several complexes containing modified residues  , whereas for AaC/UT these G4 imino signals were very broad (T strand. These structures may contain G-quadruplex motifs instead of dangling ends.When modified G-rich oligoribonucleotides were hybridized to the U pCa/UT) . In sodiry broad . These r+ cations, the migration rate of bands corresponding to modified QD/GT complexes   B.T hybrids containing a phosphate group or adenosine at the 5′-end of QD (T complexes containing non-nucleotide linkers migrated with a mobility similar to that of the model CCC/UT (T (lane 8′), Aaa/UT (lane 10′) low-intensity fluorescence bands were observed after staining with NMM  was similarly low, indicating a weak affinity of NMM towards CCC/UT. For NMM complexes with Aaa/GT and paa/GT, we observed a moderate 7- to 9-fold enhancement of NMM fluorescence emission, whereas, for Aaa/UT and paa/UT, a 4–6.5-fold enhancement of NMM fluorescence emission was determined. The intensities of the fluorescence signals of the NMM in complex with Aaa/UT and paa/UT were significantly higher compared to those obtained for NMM in complex with CCC/UT duplex. These results confirmed the selective binding of NMM to the non-canonical G-quadruplex domain of Aaa/UT and paa/UT.A comparison of the migration of QD/Gl CCC/UT . Most ofwith NMM B. To conn of NMM . The int1H NMR spectra after the addition of one equivalent of NMM to NMR samples containing paa/GT, paa/UT, or Aaa/UT  . In general, in potassium phosphate buffer, all QD/GT structures were more stable than the corresponding QD/UT complexes; their Tm values were higher at least by 6.0 °C. For paa/UT, Aaa/UT, and AaC/UT, molecules for which the presence of noncanonical G-quadruplex motifs was suggested, analysis of the melting curves at 295 nm indicated that these motifs were less stable than their QD/GT counterparts (in average by 7.25 °C). Apparent Tm values determined for the quadruplex–duplex hybrid structures in the presence of sodium cations (+ versus 160 mM Na+). By comparison, the exchange of K+ with Na+ caused a significant decrease in the stability of the G-quadruplex domain of QD/GT, which could be estimated from the melting curve recorded at 295 nm and in 1H NMR spectra (To further characterize QD/Gpossible . Howevere buffer . All QD/69.4 °C) . The onl69.4 °C) . In addi cations  were com spectra .1H NMR spectra recorded as a function of temperature. 1H NMR spectra of CCC/GT, paa/GT, and pL3C/GT obtained in the range of 25–75 °C in buffers containing 60 mM K+ and 160 mM Na+  verified the parallel topology of their G-quadruplex motifs. For QDH structures containing chemically modified G-rich strands, an increase in the intensities of the Cotton effects at 265 nm was observed compared to CCC/GT. As expected, the shape of the CD spectrum of the CCC/UT duplex with dangling ends was similar to that of the quadruplex–duplex hybrids; however, the bands at 265 nm were of lower intensity than the spectra of the corresponding QDH structures  was 2.7 °C higher than that for CCC/GT 3G3 resulted in a conversion of G-quadruplex topology from antiparallel to parallel [T and pL4C/GT quadruplex–duplex hybrids were observed in the native PAGE experiment. The formation of two conformers was also detected for the paa/GT complex containing abasic residues located in loop and junction positions. It has been previously reported that base composition proximal to the junction between duplex and G-quadruplex motifs can also play an important role in both the structure and stability of the hybrids. For example, when the terminal C:G base pair was substituted with a less stable T:A base pair in a junction, two bands appeared in gel electrophoresis, probably due to two different conformations present in solution [T relative to CCC/GT. In contrast, the presence of the non-nucleotide modifications or G4 ligand did not affect the stability of duplexes with dangling ends.Although a type of G-rich oligonucleotide (DNA or RNA) did not change the structural preferences of QD/Gx domain  in a solparallel . The staparallel . In the solution . In geneT complex. For the CCC/UT molecule, we considered the possibility of the formation of structures other than Dss. For this purpose, we used the RNAstructure software, which suggested several stable secondary structures with additional canonical Watson-Crick or non-canonical base pairs  prevented the formation of the G-quadruplex domain in the CCC/Use pairs . Howeverspectrum  indicateU tetrad . The bin binding . In summ binding ,85,86. A binding . These dwww.ncbi.nlm.nih.gov/refseq) and the IGV tool (www.broadinstitute.org/igv (accessed on 31th October 2019)), we identified potential target sequences in protooncogenes EGFR, KRAS, HER2, and genes implicated in human Mendelian dominant disorders, such as PTPN11 and FGF23. The G-rich oligonucleotides may also be considered for allele-specific targeting. For example, the well-recognized cancer-driving EGFR mutation, L858R (c.2573T>G), introduces a 5′–GGGCUGG–3′ to 5′–GGGCGGG–3′ change in the sequence of the EGFR mRNA, creating a potential target for G-rich oligonucleotides [We have recently shown that the formation of a stable G4 motif on an mRNA template was an effective steric hindrance for ribosomes and results in stalling and inhibition of protein synthesis. The silencing activity of oligoribonucleotides modified with 1,6-hexanediol and an abasic linker toward the EGFR mRNA target appeared to be at least 20% higher than for classical 16 nt antisense oligonucleotides . Severalleotides . L858R nLigands interacting with G4s have attracted significant attention as potential anticancer therapeutics ,92. In vT and UT, that differ in one nucleotide only, based on the formation of alternative structures, i.e., quadruplex–duplex hybrids or duplexes with dangling ends, respectively. We designed DNA and RNA G-rich oligonucleotides with the ability to trigger the formation of a G-quadruplex motif only when hybridized to the GT target and leaving an unstructured G-rich fragment when hybridized to the UT sequence. We also showed that the replacement of RNA G-rich oligonucleotides with DNA decreased the thermal stability of the quadruplex–duplex hybrid structure and the duplex with dangling ends. Subsequently, we demonstrated that abasic residues and aliphatic linkers can mimic the external loops of bimolecular RNA G-quadruplexes. The quadruplex–duplex hybrid structures containing these non-nucleotide modifications exhibited similar stability as their unmodified counterparts. Unexpectedly, we noticed that the presence of two abasic modifications in G-rich strands induced the formation of non-canonical G-quadruplexes after hybridization to the UT target. Finally, RNA G-rich oligonucleotides with a covalently attached carbazole derivative, o-BMVC-C3, were shown to selectively bind and stabilize a G-quadruplex domain of QDH. The obtained results advance our knowledge and ability to predict structures adopted by G-rich sequences and can be used as a starting point to design anti-EGFR G-rich antisense oligonucleotides. Moreover, o-BMVC-C3 covalently attached to an oligonucleotide allows fluorescent probe visualization of sequences containing two GGG tracts. In this article, we proposed a method to discriminate between two similar target RNA sequences, G


RNA-based therapeutics are emerging as a powerful platform for the treatment of multiple diseases. Currently, the two main categories of nucleic acid therapeutics, antisense oligonucleotides and small interfering RNAs (siRNAs), achieve their therapeutic effect through either gene silencing, splicing modulation or microRNA binding, giving rise to versatile options to target pathogenic gene expression patterns. Moreover, ongoing research seeks to expand the scope of RNA-based drugs to include more complex nucleic acid templates, such as messenger RNA, as exemplified by the first approved mRNA-based vaccine in 2020. The increasing number of approved sequences and ongoing clinical trials has attracted considerable interest in the chemical development of oligonucleotides and nucleic acids as drugs, especially since the FDA approval of the first siRNA drug in 2018. As a result, a variety of innovative approaches is emerging, highlighting the potential of RNA as one of the most prominent therapeutic tools in the drug design and development pipeline. This review seeks to provide a comprehensive summary of current efforts in academia and industry aimed at fully realizing the potential of RNA-based therapeutics. Towards this, we introduce established and emerging RNA-based technologies, with a focus on their potential as biosensors and therapeutics. We then describe their mechanisms of action and their application in different disease contexts, along with the strengths and limitations of each strategy. Since the nucleic acid toolbox is rapidly expanding, we also introduce RNA minimal architectures, RNA/protein cleavers and viral RNA as promising modalities for new therapeutics and discuss future directions for the field. The use of a synthetic oligonucleotide to alter protein expression through Watson–Crick hybridization of RNA was first described by Zamecnik and Stephenson in 1978 ,3. CurreI.in vivo and expanding the scope of disease targets that can be effectively addressed. Current ASO research can be broken down into three aims: progressing on ASO medicinal chemistry; enhancing tissue-specific targeting and delivery; and gaining a better understanding of ASO internalization into cells and endocytosis.Single-stranded oligonucleotides (ssONs) encompass three main groups of therapeutics: RNase-H active oligonucleotides, steric-blocking oligonucleotides, and aptamers. RNase-H active and steric-blocking oligonucleotides that bind an RNA target in a sequence-specific manner have been dubbed antisense oligonucleotides (ASOs). On one hand, RNase-H active oligonucleotides bind a cognate pre-mRNA or mature mRNA sequence, which triggers RNase-H binding and cleavage of the mRNA target . This meRp and Sp chiral phosphorus atoms. The PS stereochemistry is not controlled in standard solid-phase synthesis, which results in the production of stochastic, 2 n diasteroisomeric mixtures, where n is the number of chiral phosphorus atoms in the ASO sequence. Recent synthetic developments . . in vivo is a noncoding RNA produced by phi29-like bacteriophages (phages) ) 176]. . 176]. Din vitro, with some sequences capable of forming dimers, trimers, and higher order multimers . . 181]. Iultimers ,184. Theultimers ; exchangin vitro .et al. created chimeras by modifying the phi29 pRNA with a CD4-binding aptamer, an siRNA targeting survivin, or folic acid (FA) . . et al.  in mice . Notably in mice . Promisi in mice , breast  in mice , and col in mice .Figure The value of using the phi29 pRNA or its thermodynamically stable 3WJ as vectors for siRNA delivery lies in the platforms’ reported stability in the biological milieu and ability to integrate several functionalities in a single RNA structure. However, additional studies will be needed to help resolve the potential of pRNA as a clinically relevant siRNA delivery strategy. Specifically, it will be important for future studies to establish the pharmacokinetic/pharmacodynamic superiority of FA- or aptamer-3WJ-siRNA molecules over similar constructs lacking the 3WJ connector. Additionally, present engineering efforts are focused on the phi29 3WJ; phylogenetically related 3WJs with higher thermodynamic stabilities  may be bSequence-selective cleavage of RNA is important to implement therapeutic strategies that aim to reduce harmful RNA levels as well as to develop tools (e.g. restriction enzymes) that can be utilized in molecular biology. As therapeutics, gapmer ASOs and siRNAs achieve this goal by taking advantage of endogenous enzymes, RNase H and RISC, respectively. The chemical architecture of these nucleic acids therapeutics is largely dictated by the need to support enzyme action, placing considerable limitations on the chemical modifications that can be incorporated. However, if greater freedom in the design of therapeutic oligonucleotides was possible, it could potentially address some of their current challenges including addressing off-target effects and sequence and chemistry-dependent toxicity ,201. Mori.e. FANAzymes) [e.g. metal chelating groups of trivalent lanthanide ions Tm3+, Yb3+ and Lu3+ or divalent transition metals Zn2+ and Cu2+ among others, and metal-free systems based on moieties such as oligoamines or imidazole that can act as acid–base catalysts at physiological pH [As a result, there has been significant interest in the development of molecular constructs capable of catalytic sequence selective RNA degradation in the absence of endogenous enzymes. These can be purely synthetic constructs, which will be covered here, or in-vitro selected ribozyme mimics, such as DNAzymes and XNAzymes (NAzymes) , which iNAzymes) . In thisgical pH .An important aspect for the design of artificial nucleases is that single stranded RNA is more vulnerable to cleavage than double stranded RNA . The pree.g. not in any other nearby single stranded RNA species). After cleavage, the fragments are released . This can be readily achieved if the RNA scission occurs at a central part of the RNA/artificial ribonuclease complex, leaving the cleaved complex significantly destabilized and prone to dissociation [A more advanced concept consists in forcing the formation of a single-stranded bulge in the central region of the RNA by partial complementarity . The foociation . The artPhe, taking advantage of the bulge formation design to promote turnover but display complex cleavage patterns where, in addition to the expected cleavage sites, even distant RNA regions can be accessible for cleavage [The development of metal-dependent as well as metal-free artificial ribonucleases has a significant history which has been covered in detail in previous reviews . Metal-fcleavage . Such POcleavage  or ‘dualcleavage . Howevercleavage ,216.e.g. peptides) to the PNA backbone is straightforward and shown not to interfere with their activity [The most efficient oligonucleotide-based artificial ribonucleases reported to date are based on PNAs conjugated to metal ion chelates of Cu(II) or Zn(II). RNA targets forming 4-nucleotide bulges are cleaved at a single site with 20–30 min half-lives by Cu(II)-dependent PNA-neocuproine conjugates (PNAzymes) ,218. Theactivity . Moreoveactivity . Zn(II)-activity . With boactivity ,217–220.There are still several additional limitations to overcome with this technology for it to be a proper alternative to ON therapies that depend on endogenous enzymes. Delivery to cells and specific tissues is, as for all ON therapies, a major limitation. Apart from this, there are also challenges specific to oligonucleotide-based artificial nucleases that need to be overcome. Metal-ion based “molecular scissors are in general more powerful than non-metal ion-based ones, but even the former should preferably reach higher efficiency, especially for the more biocompatible Zn(II) nucleases. Metal ion-dependent systems also need a sufficient local concentration of the metal ion in the cellular environment to function. This can potentially be achieved if diseases causing significantly raised levels of metal ions are targeted. Alternatively, the use of ‘molecular scissors’ based on chelating moieties that bind the metal tightly enough to give a stable complex may be needed, but the metal ion must retain its ability to catalyse RNA scission. The relative biocompatibility of zinc makes this metal especially attractive for therapeutic artificial ribonucleases, as it can be present in micromolar concentrations and thus potentially utilized by artificial ribonucleases with a zinc binding constant in the nanomolar range ,217–220.Under physiological conditions, a complex network that includes folding enzymes, chaperones and ATP motors, controls the elimination of misfolded proteins . The twoTargeted protein degradation is an emerging protein silencing strategy, which uses small molecules ligands to knock down a protein of interest (POI) by hijacking the endogenous UPS system. PROTACs are bifunctional molecules composed of a ligand that binds the POI and a ligand that recruits the E3 ligase. Upon formation of a ternary complex target:PROTAC:E3, the POI is marked with ubiquitin for degradation by the proteasome . PROTACsTo evaluate the potential of nucleic acids based-PROTACs, it is important to contrast the advantages and disadvantages of PROTACs compared to RNA-based therapeutics. RNAi and antisense can target essential gene products and have the advantage of being active on gene families based on sequence similarities. Since their action is at the RNA level, their effect is not influenced by the protein isoform. As previously described, both technologies suffer from potential off-target effects and rely on protein turnover, thus requiring prolonged treatments. However, PROTAC is independent of regulation on DNA/RNA and can target essential proteins directly. Unfortunately, one of the major drawbacks of PROTACs is that developing a new PROTAC system is time consuming due to unpredictable structure/efficacy relationships. Although the field has recently attracted a high degree of attention, there is still a limited availability of POI tested targets and a limited number of verified E3 ligases. Proteins with catalytic activity can be successfully drugged, but most other families, such as transcription factors or RNA-binding proteins (RBPs), lack binding sites for small ligands and therefore remain challenging targets for all small molecule-based approaches, including PROTACs. As a result, it is becoming more evident that merging two very different fields, the RNAi/antisense and PROTAC to generate a new targeting tool, could extend the therapeutic capabilities of both approaches and overcome their disadvantages.The first reported single strand oligonucleotide-based PROTAC, termed RNA-PROTAC, was introduced as a proof-of-concept for the degradation of two RBPs, LIN28 and RBFOX1   have been developed in parallel to PROTACs. However, all these methods involve the manipulation of intracellular protein degradation machinery, which limit their targets to proteins that contain cytosolic domains to which ligands can bind. Bertozzi et al. developed LYsosome TArgeting Chimeras (LYTACs) , which bOligonucleotide-based technologies have been intensively growing and have taken their place as a major therapeutic platform to address unmet medical needs. It took nearly 40 years of oligonucleotide therapeutic development for them to reach clinical utility. The 2016 approval of nusinersen and eterplirsen marked an important milestone for ASO technology, quickly followed by the 2018 approval of the siRNA patisiran. Currently, 14 RNA therapeutics are approved, and many others are in clinical development, demonstrating that this technology continues to advance at a rapid pace. However, as of today, a full understanding of antisense technology is not achieved yet. Significant adverse events such as thrombocytopenia  may occuOver the last two years, already three GalNAc-conjugated siRNA drugs  have been approved and it is likely that more will be commercialized in the near future. However, terminations of nucleic acid-based therapeutics clinical trials (e.g. revusiran) demonstrate that a better understanding of the impact of chemical modifications on activity and toxicity needs to be addressed to further develop robust drugs in clinic. Furthermore, while GalNAc drugs enable effective targeting of the liver, efficient delivery to tissues beyond the liver that enables robust clinical efficacy remains to be addressed.Given the versatility of RNA properties, innovative RNA-based technologies are intensively emerging. In addition to antisense and siRNAs, novel attractive classes of RNA therapeutics  demonstrate highly encouraging outcomes to modulate gene and protein expression, opening a new repertoire of promising platforms for drug discovery. Oligonucleotide-based delivery systems such as pRNA, NA nanostructures and SNAs have been explored as well and may offer relevant platforms to deliver nucleic acids beyond hepatic cells. Even though more understanding of their mechanism and behaviours is needed, the emerging RNA-based technologies described in this review present unique therapeutic and delivery potentials and will establish a path towards expanding RNA-based applications.


O-methylation are the most abundant types of RNA modifications. They are mediated by modification guide RNAs, also known as small nucleolar (sno)RNAs and small Cajal body-specific (sca)RNAs. We used yeast and vertebrate cells to test guide activities predicted for a number of snoRNAs, based on their regions of complementarity with rRNAs. We showed that human SNORA24 is a genuine guide RNA for 18S-Ψ609, despite some noncanonical base-pairing with its target. At the same time, we found quite a few snoRNAs that have the ability to base-pair with rRNAs and can induce predicted modifications in artificial substrate RNAs, but do not modify the same target sequence within endogenous rRNA molecules. Furthermore, certain fragments of rRNAs can be modified by the endogenous yeast modification machinery when inserted into an artificial backbone RNA, even though the same sequences are not modified in endogenous yeast rRNAs. In Xenopus cells, a guide RNA generated from scaRNA, but not from snoRNA, could induce an additional pseudouridylation of U2 snRNA at position 60; both guide RNAs were equally active on a U2 snRNA-specific substrate in yeast cells. Thus, post-transcriptional modification of functionally important RNAs, such as rRNAs and snRNAs, is highly regulated and more complex than simply strong base-pairing between a guide RNA and substrate RNA. We discuss possible regulatory roles for these unexpected modifications.In eukaryotes, rRNAs and spliceosomal snRNAs are heavily modified post-transcriptionally. Pseudouridylation and 2′- O-ribose methylation and pseudouridylation of other RNA molecules. Their specificity is determined by the so-called antisense elements (ASEs)—short sequences complementary to substrate RNAs. In the case of 2′-O-methylation, ASEs are located upstream of the D/D′ box (CUGA) motif in box C/D snoRNAs; exactly the fifth nucleotide from the D-box sequence determines the position of 2′-O-methylation in a substrate RNA. Pseudouridylation is mediated by box H/ACA snoRNAs. The ASEs in this type of modification guide RNA are formed by internal loops in 5′ and 3′ stems associated with box H (ANANNA) and box ACA, respectively; these loops are usually called pseudouridylation pockets. Typically, the distance from the box H or box ACA to the position of the target uridine inside the pocket is 15 ± 1 nt  are abundant noncoding nuclear RNAs. The vast majority of these RNAs function as guide RNAs to mediate site-specific post-transcriptional 2′-O-methylation are the most abundant and functionally important post-transcriptional modifications in eukaryotic rRNAs and spliceosomal snRNAs . Yet, in a later study SNORA24 activity on this position was not confirmed using yeast nuclear extract , shows complementarity to 18S rRNA at position 1607   B. Yet, n18S rRNA . To testXenopus, Pus7p  nor the equivalent position in yeast 18S rRNA (yeast Pus7p substrate) is modified.We screened several yeast mutant strains deficient for different pseudouridine synthases and found that yeast Pus7p was responsible for this modification B. It is s, Pus7p , differ s, Pus7p D. UltimaX. tropicalis SNORA15 . Here again, no modification is found at the predicted target position in 18S rRNA from any species. Although the 18S rRNA sequence surrounding this position is extremely conserved, overexpression of xtSNORA15 in yeast cells did not induce modification of yeast 18S rRNA at position 1136. At the same time, when we expressed the corresponding artificial substrate RNA alone as a negative control, it became modified. That is, the endogenous yeast modification machinery recognizes and modifies this substrate RNA, but not the same sequence within endogenous yeast 18S rRNA. Unfortunately, in this case we could not identify the corresponding yeast guide RNA or stand-alone pseudouridine synthase, nor could we further test xtSNORA15 activity.The modification of the SNORA24 substrate RNA by yeast Pus7p is not the only case in which the endogenous modification machinery is active on an artificial substrate RNA but not on endogenous yeast rRNA. Another example is a potential substrate for one of the two copies of xtSNORA24 and yeast Pus7p, we identified several other vertebrate snoRNAs that were functional in vivo on artificial substrate RNAs, but not on endogenous rRNAs. One such snoRNA is the orphan vertebrate SNORA18. It has an evolutionarily conserved ASE that can base-pair with 18S rRNA to convert U1720 to pseudouridine . These growing conditions did not induce modification activity of the yeast optimized SNORA18 on yeast 18S rRNA at position 1656 . X. laevis SNORA28 contains the corresponding ASE, yet we observed the lack of this modification in X. laevis 18S rRNA. In fact, this ASE is somewhat imperfect in xlSNORA28 , correlated with the environmental conditions in which these species normally live  could induce pseudouridylation of Xenopus U2 snRNA at position 60 . Besides, these additional interactions might allow substrate RNA to slide easier into an alternative configuration of a functional modification guide RNA domain. That is what we presumably observed in the cases of vertebrate U92/SCARNA8 activities on U2 snRNA  and SNORD48  complementary regions are overlapping. This might be the reason that 28S-Cm1868 is only fractionally modified by SNORD48 . SimilarxlSNORA28 on 18S rRNA. On the other hand, some other types of stress might be involved in induction of RNA modifications mediated by imperfect yet potentially functional guide RNAs. In fact, in yeast U2 snRNA only one out of two inducible pseudouridines is formed in response to heat shock, and this isomerization is catalyzed by stand-alone pseudouridine synthases Pus7p; the other, snoRNA-dependent, inducible pseudouridine forms in response to nutrient stress  deficient for factors involved in 18S nonfunctional rRNA decay, or NRD (xtSNORA15) was not detected (data not shown). These negative results do not exclude the possibility that a novel 18S NRD-independent pathway of 18S rRNA quality surveillance might be triggered. This line of rRNA modification research is worth pursuing.Another possibility is that these modifications occur only on mutated, misfolded or misassembled RNA. These abnormally modified RNA molecules might be targeted for degradation and therefore undetectable. Since we predicted and analyzed unusual pseudouridine synthase activities on 18S rRNA, we chose several yeast strains . Selective synthetic media containing either glucose or galactose as a sugar source were used to grow yeast cells. Heat shock conditions were 37°C for yeast and 29°C for amphibians.To express test RNAs ectopically, the listed constructs were introduced into yeast and X. tropicalis pseudouridine synthase Pus7 was amplified from total RNA using the OneTaq One-step RT-PCR kit (New England Biolabs). XmaI and XhoI restriction sites were added with oligonucleotides and the resulting fragment was cloned into the p415Gal1 vector.The annotated protein coding sequence for RNA was extracted from control and transfected cells using the TRIzol reagent in the case of vertebrate cells or hot acid phenol in the case of yeast cells. RNA was purified using the Direct-zol RNA miniprep kit (Zymo Research). Proper processing and expression levels of exogenous RNAs were analyzed using Northern blotting as described .O-methylated residues, we used fluorescent primer extension-based techniques as described (O-methylated positions. 6-FAM-labeled oligonucleotides specific for yeast and vertebrate U2 snRNA, 18S rRNA, and U87-based artificial substrate RNAs were described earlier (To detect pseudouridines and 2′-escribed . RNA tre earlier . 6-FAM-lSupplemental material is available for this article.


Knowing chromatin components at a DNA regulatory element at any given time is essential for understanding how the element works during cellular proliferation, differentiation and development. A region-specific chromatin purification is an invaluable approach to dissecting the comprehensive chromatin composition at a particular region. Several methods  have been developed for isolating and analyzing chromatin components. However, all of them have some shortcomings in identifying non-coding RNA associated with DNA regulatory elements.N-methyl pyrrole (P)-N-methylimidazole (I) (PI) polyamide probe, which binds to a specific sequence in double-stranded DNA via Watson–Crick base pairing as a minor groove binder. This new technique is called proteomics and RNA-omics of isolated chromatin segments (PI-PRICh). Using PI-PRICh to isolate mouse and human telomeric components, we found enrichments of shelterin proteins, the well-known telomerase RNA component (TERC) and telomeric repeat-containing RNA (TERRA). When PI-PRICh was performed for alternative lengthening of telomere (ALT) cells with highly recombinogenic telomeres, in addition to the conventional telomeric chromatin, we obtained chromatin regions containing telomeric repeat insertions scattered in the genome and their associated RNAs.We have developed a new approach for affinity purification of specific chromatin segments employing an PI-PRICh reproducibly identified both the protein and RNA components of telomeric chromatin when targeting telomere repeats. PI polyamide is a promising alternative to simultaneously isolate associated proteins and RNAs of sequence-specific chromatin regions under native conditions, allowing better understanding of chromatin organization and functions within the cell.The online version contains supplementary material available at 10.1186/s13072-021-00421-8. Genome activities, such as transcription and replication, are often achieved by DNA regulatory elements interacting with protein complexes and modified histone proteins to shape numerous and unique chromatin landscapes , 2. ReceKnowing all the players acting at DNA regulatory elements is a major step toward understanding how the element works. Several approaches are available for isolating and analyzing chromatin at specific regions . We focuN-methyl pyrrole (P)-N-methylimidazole (I) (PI) polyamides for the visualization of telomeres and the assessment of telomere length  at 55 °C for 1 h. The prehybridized coverslips were then incubated with hybridization solution  at 55 °C overnight. After hybridization, the coverslips were washed twice with wash buffer  at 55 °C for 30 min twice. To remove non-specific binding of RNA probe, cells were incubated in RNase A buffer  for 1 h at 37 °C. After RNase A treatment, cells were washed with wash buffer2  and wash buffer3  for 30 min at 55 °C. For detection, coverslips were washed in TBST  for 5 min at room temperature, incubated with blocking solution [1 × Blocking reagent  in TBST] for 5 min at room temperature, and then incubated with anti-DIG Rhodamine  and 15 nM Silicon Rhodamine (SiR)-TH59 for 90 min at room temperature. After staining with 0.5 μg/mL DAPI in TBST for 5 min at room temperature, unbound antibodies were removed by washing three times with TBST for 5 min. Coverslips were then mounted and image acquisition was performed with a DeltaVision microscope. Signal intensities were measured by Plot Profile tools in Fiji and line plots were created using R software [Telomere staining with RNA-FISH was performed following an RNA-FISH method combined with immunofluorescence as described in . BrieflyAdditional file 1: Figure S1. Structure of TH59-DB and enrichment of ncRNAs by PI-PRICh within subtelomeric regions of MEL cells. a Chemical structure of TH59-DB. Py, N-methyl pyrrole (blue) and Im, N-methyl imidazole (red). b A structural model of TH59-DB binding to DNA. c, ncRNAs mapped to the region near the end of each chromosome  from Experiment 1. A genomic view of the terminal regions of the q arm of each chromosome based on the mouse reference genome, GRCm38 (mm10). RNAs were mapped to the terminal region of each chromosome. Results with the input, masked TH59-DB and TH59-DB are shown.Additional file 2: Figure S2. Comprehensive identification of telomeric chromatin-associated RNAs in MEL cells (Experiment 2). a The percentage (yellow) of telomeric repeat reads, including TERRA transcripts, in the input and TH59-DB pull-down fractions from MEL cells. The number of single reads, including (TTAGGG)5 or (CCCTAA)5, extracted from input and TH59-DB pull-down fractions were divided by the total number of reads. The gray color depicts other reads. b Scatter plot of fragments per kilobase of per million mapped reads (FPKM) of the TH59-DB pull-down fraction versus that of the input sample for each RNA. The telomerase RNA component, TERC (most enriched in the TH59-DB pull-down fraction), is highlighted in blue. RNAs enriched more than 100-fold in the TH59-DB pull-down fraction are plotted below a red dotted line. c Bar graph of FPKM of telomerase RNA-component (TERC) in input and TH59-DB pull-down fractions. d Sequence analysis of DNA fragments captured by TH59-DB telomeric repeat reads. The percentage (yellow) of telomeric repeat reads including TTAGGG or CCCTAA, in the input and TH59-DB pull-down fractions from MEL cells. Note that results are similar to those shown in Fig. Additional file 3: Figure S3. Genomic PCR to test telomeric sequence insertion. a A scheme of genomic PCR to test telomeric repeat insertion into the flanking regions of SPOCK3, USP16, CKS1B, NRDC, PAM and KIAA1671 introns. Primer sets and expected PCR products are shown. b PCR products of each intron amplified by the three primer sets with HeLa1.3 and ALT U2-OS genomic DNA. Lanes 1–3 and lanes 5–7 for SPOCK3, lanes 8–10, and lanes 12–14 for USP16, lanes 15–17 and lanes 19–21 for CKS1B, lanes 22–24 and lanes 26–28 for NRDC, lanes 29–31 and lanes 33–35 for PAM, lanes 36–38 and lanes 40–42 for KIAA1671. 100 bp or 1 kb ladder was loaded at the center lane of the gel as a size marker (M).Additional file 4: Figure S4. PI-PRICh identifies ALT cell-specific ncRNAs transcribed from around the inserted telomeric repeats. Telomeric repeat-associated ncRNAs mapped to intron regions of CKS1B, NRDC, PAM and KIAA1671 genes in ALT U2-OS cells. These ncRNAs were highly enriched in the TH59-DB pull-down fraction of U2-OS cells, but not in HeLa1.3. The positions of the inserted telomeric repeats are indicated by red arrowheads.Additional file 5: Figure S5. DNA sequence analysis of TH59-DB binding sites. Genomic regions coding the introns of CKS1B, NRDC, PAM, and KIAA1671 were specifically enriched in the TH59-DB pull-down fraction from ALT U2-OS cells. These intron regions were enriched in the TH59-DB fraction from U2-OS, but not in HeLa1.3 cells. The positions of the inserted telomeric repeats are indicated by red arrowheads.Additional file 6: Figure S6. RNA-FISH for intronic ncRNAs identified by PI-PRICh in ALT cells and a model for chromatin tethering to telomeres with ncRNAs. a RNA-FISH for the USP16 intron and the simultaneous telomere labeling with the fluorescent TH59 probe. First column, DAPI signal; second column, ncRNA signal of TERRA; third column, fluorescent TH59 signal (telomere); fourth column, merged image of DAPI (blue), ncRNA (green) and telomere (red). b Enlarged images of the boxed region in a. Line plot of the USP16-intronic RNA signal and the telomeric signal on the white dotted line in the left merged image. c Bar graph of percentages of cells in which USP16, SPOCK3, CKS1B and NRDC intronic RNA signals were co-localized with telomere signal. Three independent experiments were performed and the numbers of ncRNA signals measured for U2-OS cells were from 100 cells for each experiment. Error bars show standard deviation. d A model of how the genome region with inserted telomeric repeats tethers to telomere in ALT cells.Additional file 7: Table S1. Mass spectrometry data of PI-PRICh experiments from MEL cells.Additional file 8: Table S2. Mapped and counted RNA fragments from MEL cells. Sheet 1: RNAs with over 100-fold enrichment in TH59-DB fraction by PRICh in MEL experiment 1. Sheet 2: A non-filtered result of counted RNA fragments in MEL experiment 1. Sheet 3: RNAs with over 100-fold enrichment in TH59-DB fraction by PRICh in MEL experiment 2. Sheet 4: A non-filtered result of counted RNA fragments in MEL experiment 2.Additional file 9: Table S3. RNAs enriched in the U2-OS or HeLa1.3 cell line by filtering criteria. Sheet 1: U2-OS enriched RNAs by filtering criteria. Sheet 2: HeLa1.3 enriched RNAs by filtering criteria.Additional file 10: Tables S4. Filtration procedure to identify RNAs enriched in the U2-OS or HeLa1.3 cell line. RNAs in U2-OS experiment 1. Sheet 1: Non-filtrated count data. Sheet 2: Enriched more than 25-fold. Sheet 3: 50 times more than another cell line.Additional file 11: Tables S5. Filtration procedure to identify RNAs enriched in the U2-OS or HeLa1.3 cell line. RNAs in HeLa1.3 experiment 1. Sheet 1: Non-filtrated count data. Sheet 2: Enriched more than 25-fold. Sheet 3: 50 times more than another cell line.Additional file 12: Table S6. Filtration procedure to identify RNAs enriched in the U2-OS or HeLa1.3 cell line. RNAs in U2-OS experiment 2. Sheet 1: Non-filtrated count data. Sheet 2: Enriched more than 25-fold. Sheet 3: 50 times more than another cell line.Additional file 13: Table S7. Filtration procedure to identify RNAs enriched in the U2-OS or HeLa1.3 cell line. RNAs in HeLa1.3 experiment 2. Sheet 1: Non-filtrated count data. Sheet 2: Enriched more than 25-fold. Sheet 3: 50 times more than another cell line.Additional file 14: Table S8. Mouse subtelomeric and telomeric regions for FPKM calculation in Fig. Additional file 15: Table S9. Primers for telomeric insertion test and for RNA FISH and DNA sequences to search targets for the grep command in the UNIX system.


ORF1a to ORF1b is essential for propagation of coronaviruses. A combination of genomic features that make up PFE—the overlap between the two reading frames, a slippery sequence, as well as an ensemble of complex secondary structure elements—puts severe constraints on this region as most possible nucleotide substitution may disrupt one or more of these elements. The vast amount of SARS-CoV-2 sequencing data generated within the past year provides an opportunity to assess evolutionary dynamics of PFE in great detail. Here we performed a comparative analysis of all available coronaviral genomic data available to date. We show that the overlap between ORF1a and b evolved as a set of discrete 7, 16, 22, 25, and 31 nucleotide stretches with a well defined phylogenetic specificity. We further examined sequencing data from over 350,000 complete genomes and 55,000 raw read datasets to demonstrate exceptional conservation of the PFE region.The programmed frameshift element (PFE) rerouting translation from  ORF1ab encoding nsps essential for coronaviral life cycle. As the designation “ab” suggests it contains two reading frames with the 3’-end of ORF1a overlapping with the 5’-terminus of ORF1b. ORF1b is in −1 phase relative to ORF1a and translated via the −1 programmed ribosomal frameshifting controlled by the programmed frameshift element (PFE). As ORF1b encodes crucial components of coronavirus transcription/replication machinery including the RNA-dependent RNA polymerase (RdRp) disrupting PFE abolishes viral replication completely  it is 23 nt  mpletely . PFE conmpletely . The seqmpletely . Becausempletely ) revealimpletely . In addiis 23 nt .ORF1a and ORF1b overlap is phylogenetically conserved. It evolved in a stepwise manner, where the changes in the overlap length are results of the loss of ORF1a stop codons leading to ORF1a extension, and the acquisition of insertions and deletions causing early stops of ORF1a.Our group has been interested in the evolutionary dynamics of overlapping coding regions . The vaspro, HEL, M, and N proteins suggested that γ- was more closely related to δ-coronavirus, while α- and β-coronavirus cluster together forming a distant clade .The 35,152 coronaviral entries in the NCBI taxonomy database were sorted by length, and only those larger than 14,945 nt were kept, leaving a total of 4,939 genomes. The slippery site and following overlap sequences were manually inspected, in case that the slippery site was incorrectly annotated. We further filtered out those entries if they contain no annotation information, or have gapped sequences in the overlap. By applying these filters, we finally had 4,904 coronavirus entries (Table S1), of which the overlap could be unambiguously determined.δ-coronavirus entries in Table S1, the first 13 amino acids of ORF1b were taken to generate a consensus sequence using WebLogo  and then subdivided into ten regions based on CDS features: ORF1a (including nsp10), ORF1b (starting with nsp12), S, ORF3a, E, M, ORF6, ORF7a, ORF8, N, and ORF10. For each region, we scanned and discarded sequences containing too many ambiguous nucleotides to remove data with too many sequencing errors. Thresholds were 0.5% for the S gene, 0.1% for ORF1a and ORF1b genes, and 1% for all other genes. We mapped individual sequences to the NCBI reference genome (NC_045512) using a codon-aware extension to the Smith-Waterman algorithm implemented in HyPhy  translat


Pseudomonas capeferrum TDA1 based on RNAzol® RT, glycogen and a final cleaning step. It yields a high-quality RNA from cells grown on TDA1 and on phenol compared to standard assays conducted in the study. To our knowledge, this is the first report tackling the problem of polyphenolic compound interference with total RNA isolation in bacteria. It might be considered as a guideline to improve total RNA extraction from other bacterial species.Bacterial degradation of xenobiotic compounds is an intense field of research already for decades. Lately, this research is complemented by downstream applications including Next Generation Sequencing (NGS), RT-PCR, qPCR, and RNA-seq. For most of these molecular applications, high-quality RNA is a fundamental necessity. However, during the degradation of aromatic substrates, phenolic or polyphenolic compounds such as polycatechols are formed and interact irreversibly with nucleic acids, making RNA extraction from these sources a major challenge. Therefore, we established a method for total RNA extraction from the aromatic degrading  Pseudomonas species are by far the best studied ones because of their unique properties to degrade and tolerate a wide variety of xenobiotic compounds [During several decades, various microorganisms have evolved metabolic pathways to degrade environmental pollutants derived from anthropogenic activities  which are present in different habitats . In natuompounds ,7.ortho- or meta-cleavage pathway. During ortho-cleavage pathway, the aromatic ring fission occurs between two hydroxyl groups while during meta-cleavage this is done between one hydroxylated carbon and other adjacent non-hydroxylated carbon. Both ways are catalyzed by intradiol and extradiol dioxygenases, respectively, using Fe+3 and Fe+2 at the active site [Pseudomonas and other bacterial species [The mayor challenge in bacterial degradation is to overcome the resonance energy that stabilizes the aromatic ring ,9. To doive site ,12. Finaive site –16. Such species . Several species –21. A mo species .Nucleic acid isolation is regularly the starting point for all downstream applications. However, isolation of intact RNA can be a challenge due to several factors including hydrolysis susceptibility, enzymatic and heat degradation . To overIn many follow up applications including cDNA library construction, gene expression studies and next generation sequencing, the reproducibility and validity of the data depend on the quality of the RNA extracted . In addiPseudomonas capeferrum TDA1 growing on three different carbon sources including phenol, succinate and 2,4-diaminotoluene ; an aromatic diamine and precursor for the production of polyurethane. In previous reports, this compound was degraded by Pseudomonas capeferrum TDA1 and a preliminary degradation pathway was suggested [In this study, total RNA extraction was conducted for the uggested ,29. Regauggested –34. In oPseudomonas capeferrum TDA1 was cultivated in Hartman’s mineral salts medium [3 (10 mM) while the supernatant was discarded.Prior to the experiment, s medium  and succ560 = ~0.8). The cells grown on phenol were harvested after 5 days and added to a fresh medium, reaching the exponential phase 4 days later. After that, two milliliters of the culture of every single media were centrifuged , re-suspended in RNA Later  and stored at -80°C.The pellets were added to mineral media containing only one carbon source  and incubated for 8 hours (succinate) and 7 days  until they reached the exponential phase , peqGOLD TriFast  and phenol chloroform protocol [® RT  with the following modifications: Initially, the cell solution was centrifuged  to collect cells and discard the supernatant. 0.5 milliliters of RNAzol® RT were added to the pellets and re-suspended in the reagent. Each solution was transferred to the lysing matrix B tubes and homogenized using the FastPrep-24  during 35 seconds at 6.5 m/s. After homogenization, the samples were transferred to 1.5 mL micro-centrifuge tubes and 0.2 mL of RNase-free water were added for DNA, protein, and polysaccharide precipitation according to the protocol [The following standard laboratory kits and methods were tested for RNA extraction from protocol . Additioprotocol .g during 2.5 minutes at room temperature. The supernatant was removed carefully and the pellets were solubilized by adding RNase free water (45 μL)  following the protocol suggested by the manufacturer for total RNA extraction. Due to the low RNA concentration (2.5–3.0 ng/μL) yielded from 2,4-TDA samples, a pooling step (2 or 3 samples per pool) was added to the protocol before the cleaning up process.Afterwards, the supernatant was transferred to a new 1.5 mL low binding micro-centrifuge tube with an equal volume of isopropanol and 1 μL of glycogen . The samples were incubated at −80°C for 40 minutes and centrifuged at 12,000 g for 10 minutes at room temperature. The RNA pellets were washed twice with 0.4 mL of 70% ethanol (v/v) and centrifuged at 8,000  (45 μL) . Finally260/280 and A260/230 values for RNA samples were measured using Nanodrop ND-1000 Spectrophotometer . The quantification of RNA was done in triplicates. After RNA concentrations in the samples were analyzed, RNA integrity was determined in 1 μl of total RNA using the RNA Nano (succinate and phenol) and Pico chips  assays and Agilent 2100 Bioanalyzer  in accordance with the manufacturer’s protocols. Samples with an integrity number (RIN) above 7.0 were selected for further downstream applications.Total RNA was quantified using a fluorescent RNA-binding dye Qubit Fluorometer  according to the manufacturer’s instructions. APseudomonas capeferrum TDA1 growing on aromatic compounds  and succinate.Routine molecular applications such as RT-PCR, NGS, RNA-seq require RNA with high purity and integrity ,26. At pFirst, two commercial kits  based on spin columns following the manufacturer’s recommendations yielded low RNA quantity for phenol (ranged from 5.3 to 18.9 ng/μL) and 2,4-TDA samples (ranged from 2.0 to 2.1 ng/μL), compared to RNA isolated from cells grown on succinate that showed higher concentrations for both protocols.P. capeferrum TDA1 grown on succinate, phenol and 2,4-TDA , RNeasy power plant kit  and modified RNAzol RT  method were effective inhibiting protein and phenol contamination [Apure RNA ,40. Tablmination ,40.260/A230 ratio is a sensitive indicator of contaminants such as: guanidine thiocyanate (GTC), guanidine hydrochloride (GuHCl), EDTA, polysaccharides and other secondary metabolites. The RNeasy kit , RNeasy power plant kit  and modified RNAzol RT  showed values that correspond to high RNA purity (260/A280 (≤ 1.85) and A260/A230 (≤ 1.65) ratios for succinate and phenol samples, which indicates organic contamination that compromises the RNA quality [The AA purity . On the  quality ,41,42. F quality .260/A280 and A260/A230 ratios out of the acceptable range, suggesting possible problems in the extraction due to the presence of polyphenolics, polysaccharides and secondary metabolites that precipitated with the nucleic acids [RNA isolation of cells grown on 2,4-TDA using spin columns presented Aic acids .These results are consistent with previous reports which demonstrated that commercial kits using spin columns are not suitable for RNA extraction from plants rich in polysaccharides and polyphenols. Phenolic substances reduce the efficiency of the column and can bind irreversibly to proteins and nucleic acids, leading to degradation and subsequent low-quality RNA ,26,44. TPseudomonas capeferrum TDA1 reveals the presence of the gene yfiH encoding for a polyphenol oxidoreductase laccase (EC 1.10.3.2) (data not showed). This enzyme oxidizes a broad range of phenolic and non-phenolic compounds and has been isolated from several Pseudomonas species [However, not all RNA yields from cells grown on aromatic compounds had the same result. In the case of 2,4-TDA, the poor RNA yield and quality demonstrated protein and organic contamination. During incubation, a browning effect in the media was observed, which suggests the presence of PPO enzymes catalysing the oxidation of diphenols to quinones ,46 that  species . In this species . Previou species –51, howeP. capeferrum TDA1. RNA extracted from cells grown on phenol presented low A260/A280 and A260/230 ratios (Methods (phenol/chloroform and TriFast) based on guanidinium thiocyanate–phenol–chloroform were less efficient for the isolation of RNA from 0 ratios  for bothIn the case of 2,4-TDA samples, the phenolic compounds and secondary metabolites in the oxidized form could interfere with the RNA yield. Negligible quantities of RNA extracted from plant tissues was reported earlier using a reagent based on guanidinium thiocyanate . Also, iFurther analysis of RNA integrity using an Agilent 2100 Bioanalyzer showed RIN values ≥ 8.50  for RNA On the other hand, RNA isolation from succinate samples  revealed RIN values ≥ 7.90, which confirms the complexity of high-quality RNA extraction from cells grown on aromatic compounds. In contrast, the phenol/chloroform protocol obtained low RIN values (≤ 5.20) for all the samples.260/280 and A260/230 ratios out of the acceptable range for high-quality RNA (lower than 1.8 or over 2.0) and a RIN over 8. This finding is compatible with other studies which propose that RNA purity and RNA integrity are unrelated and there is no significant correlation between them [RNA samples (succinate and phenol) extracted with the TriFast method exhibited Aeen them ,57. Howeeen them , transcreen them , differeeen them  that intAs it has been discussed above, to obtain high-quality RNA represents a fundamental step for high technology platforms including NGS that have provided many valuable insights into biological systems.® RT as a single step procedure removed DNA contamination without DNase treatment and reduced RNA time handling and helped diminished sample degradation, as has been tested in similar methods [The modified protocol presented in this study demonstrated an increase in RNA yield and quality from all the carbon sources  compared methods .Gyrodactylus salaris samples was useful for increasing total RNA quality and yield [® RT [Regarding the cells grown on 2,4-TDA, pooling was an important key in the modified method. Pooling samples may provide a solution when RNA input is insufficient in a single sample for subsequent analyses . A prevind yield . Moreovend yield . Considend yield . Glycogend yield ,68. Finald [® RT . This fild [® RT –72.Pseudomonas capeferrum TDA1 grown on succinate, phenol and 2,4-TDA were compared. Conventional methods failed to yield high quality RNA from cells grown on 2,4-TDA Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.


A compact and efficient IC architecture is presented as an alternative to laser-trimmed precision thin-film resistors or look-up tables. The objective is to keep the device, such as a four-terminal Wheatstone bridge, but to compensate for post-manufacturing offset and to avoid the so-induced degradation of performances in terms of full-scale, non-linearity, power supply noise rejection and scale factor. Expected advantages are a reduced cost due to the electrical-only implementation and a possible on-field calibration of high-end sensors. Application of the proposed solution is illustrated on an example to demonstrate improvement factors on offset and sensitivity accuracy of 32 and 10, respectively. Additionally, the power supply rejection ratio is improved by 30 dB. The experimental results finally demonstrate both efficiency and versatility of the proposed solution thanks to a first silicon prototype, fabricated in a 0.35 μm Technology from AMS, connected to an off-the-shelf pressure sensor. Nowadays, MEMS-based or micromachined resistive sensors have become widely used in many fields of application such as automotive, medical, defense, space, etc. While their consumer counterparts may afford being affected by process- and mismatch-induced scatterings, high-end resistive sensors require precise control of offset, temperature drift and scale factor after post-manufacturing calibration. Classically, a resistive sensor delivers an electrical signal at the output of a Wheatstone bridge. This simple and cheap conditioner is efficient in the absence of mismatch between resistances composing the bridge. However, microfabrication and MEMS-based manufacturing induce an offset, which directly impacts Power Supply Rejection Ratio (PSRR) and thus the minimum detectable signal . Offset The first solution consists of using a set of laser-trimmed resistors connected in series and in parallel with Wheatstone bridge resistors ,3,4,5. AThe second solution consists of using a more complex conditioner to convert the physical magnitude into a frequency, a PWM or a digital signal ,7,8. OncThis paper proposes a smart and cost-effective alternative to laser trimming based on digital potentiometers ,10,11. IIn this paper, we extend our preliminary proposal  with in-The paper is organized as follows. First, intrinsic performance limitations introduced by mismatches due to manufacturing are presented for a bare resistive sensor in a Wheatstone bridge conditioner. bV1 and bV2 are connected to the power supply voltage ddV, whereas gndV1 and gndV2 are connected to the ground (gnd). Depending on the sensor architecture, one to four of the WB resistances are sensitive to the physical input, while others are reference resistors with fixed values. Some non-exhaustive examples are:R1− = R2− = R2+ = R0 and R1+ = R0 + ΔR;Sensors with a single sensitive element: R1− = R2+ = R0, R1+ = R0 + ΔR and R2− = R0 − ΔR;Differential sensors: R2+ = R+1 = R0 + ΔR and R1− = R2− = R0 − ΔR.Full bridge sensors: where R0 and ΔR are, respectively, the nominal value of resistance and the variation of sensitive resistances induced by the physical magnitude to be measured.The Wheatstone bridge (WB) is the most common conditioner for resistive sensors. Sensor resistors are typically organized as depicted in α is the number of sensitive resistors in the bridge, i.e., 1, 2 or 4 in most of the cases. From this equation, the advantage of a WB arrangement is obvious: the differential output voltage is equal to zero when the physical signal is null, thus insuring a good power supply rejection ratio (PSRR) and a high resolution only limited by the signal-to-noise ratio of the bridge. Additionally, if the four resistances of the WB exhibit the same temperature behavior, temperature effects are cancelled.The small-signal differential output voltage of the bridge is then given by:However, process mismatches, during sensor manufacturing, lead to an unbalanced bridge in the absence of input signal. Post-fabrication calibration is then often required for high-end sensors to cancel those random effects on each sensor. This is classically implemented using an additional die with resistances that may be laser trimmed and placed in series or in parallel with WB resistors. Our proposal, illustrated in R0. Monte Carlo (MC) simulations have been performed using Cadence-Virtuoso© design environment taking into account typical mismatches for a standard microfabrication process, i.e., a gaussian distribution of mismatches with σ = 0.23% affecting WB resistors independently. It is worth noting that process variations that affect all four resistances identically do not impact WB offset (Equation (1)). Under a 5 V power supply voltage, a worst-case offset of less than 18 mV is observed. The so-obtained maximum input-referred offset corresponds to 0.36% of signal for a full bridge. Even if this offset lies in the specification of the considered application, it also induces a strong degradation of PSRR as illustrated in Let us first put in evidence limitations of a bare WB. As a conclusion for this preliminary study, small signal detection in a WB is limited by offset and PSRR. State of the art solutions have been presented in the introduction and our proposal consists in adding a compact IC inside the sensor package to allow a fully electrical calibration procedure. This will allow the so-obtained smart-sensor, after in-factory calibration, to behave like a classical passive Wheatstone Bridge in terms of output signals and external connections without offset-induced limitations.o+V, o−V, ddV and gnd), while few control inputs are used only during post-fabrication calibration. The proposed solution consists in connecting sensor resistors to a calibration ASIC as depicted in ddV and both terminals of the Wheatstone bridge as depicted in The straightforward idea, to compensate for process-induced offset, is to connect a digital potentiometer between C0 to nC) to compensate for post-fabrication offset. The calibration procedure presented hereafter reduces the impact of the on-state resistance of MOS transistors and is easy to implement:outV is measured in absence of signal.Initially, all transistors are in the on-state; differential output outV is positive (respectively negative), a resistance must be serially added to R1− (resp. R1+). The switch controlled by C0 (resp. nC) is opened to add osR1 (resp. osnR) in the path to ddV.If outV sign is obtained. The configuration code that gives the best offset can then be chosen between the first before the offset sign changes and the first after the sign changes. The maximum offset is then reduced by a factor 2n, where n is the number of compensation resistors osR.Switches are successively opened from left to right (resp. right to left) until a change of There are different ways to control switches  must be added in one branch or the other depending on the offset sign. If this resistance is discretized in 7 elements (n = 7), then each elementary resistance oRs must be equal to about 0.21% of R0. As an example, let us try to compensate the offset observed in n) and a reduction of standard deviation from 5.7 mV down to 766 µV. PSRR is also improved to increase, in the worst case, from 48 dB up to 70.5 dB. It is worth noting that PSRR remains stable over a large band of frequencies. Obviously, increasing the number of elementary resistors may increase performance in terms of residual offset and PSRR with a linear impact on the calibration time.Monte Carlo simulations are then performed to verify efficiency and results, reported in R0, and compensation resistances, osR, will have different TCR and, as illustrated in a typical example RsRp=sR can be easily calculated to compensate half of the initial offset. The so-obtained value is given by:The value of pR is calculated from Equation (2) and compensates for the remaining half of the initial offset. Then, sR and pR is nonlinear. Therefore, if sR is a linear digital potentiometer, pR cannot be controlled linearly. Second, for a sensor with a small offset, the required value of sR to compensate half of the offset is very small  and (4), Let us assume that a serial resistance is added in both WB’s branches which value is given by:With pR, obtained for k = 0, is reduced by two orders of magnitude with respect to the original implementation [opR from 100opR down to (100 − n) opR. In addition, the control of the two additional digital potentiometers, namely A and B on iA, iB and iC are generated from the same control word. Indeed, for both branches of the WB, when k elementary osR are added serially, opkR have to be removed in parallel. In the example of pR resistors are implemented as two digital linear potentiometers in parallel with R2− and R2+ and they can be linearly adjusted from 100opR down to 93opR. Finally, all three potentiometers A, B and C are controlled by a single digital word of four bits that allows them to encode 15 possible configurations.Therefore, the maximal value of entation . This isosR1 is added  in series with bV1 (resp. bV2), one opR is removed from potentiometer B (resp. A); this procedure is repeated until the offset sign changes. The procedure of offset compensation is then modified as follows: initially, all switches of potentiometers A and B are opened, and switches of potentiometer C are closed. When Electrical simulations of the modified architecture have demonstrated the same level of both offset reduction and PSRR improvement as presented in C and connected between terminals gndV1 and gndV2. Depending on application, auto-zeroing process may start when power supply is ramping-up, if a zero-input is then guaranteed, or when a specific input is set, if an independent auto-zero procedure is required. The proposed procedure involves a digital finite-state machine (FSM) controlled by the sign of the sensor differential output that sweeps all combinations and stops when offset sign has changed.In addition to the previously presented offset compensation circuitry, an automatic offset calibration procedure is added to compensate for residual offset and to ensure long sensor life by finely adjusting offset at each power-up of the circuit or upon request depending on the application. This compensation is based on a digital potentiometer similar to potentiometer n resistors, FR1…FnR, and n + 1 control switches (D0…nD). Note that resistance of each elementary FiR is small compared to osR, typically osR = FinR. Therefore, the impact of this new serial resistance on TDO is negligible. The automatic procedure uses an on-chip comparator to determine offset sign (positive or negative). Obviously, a comparator with a very low offset and a small thermal drift is required. Back to the on-going example of application, the remaining offset after coarse tuning lies in the range of ±1.55 mV (FR resistors (controlled by four bits) and an ideal comparator. Note that the theoretical improvement of the offset is only n  and a pair of resistors, R1 and R2, equal to 615 kΩ. M3 sets the bias current of the amplifier by copying the current provided by the branch of M4, M5 and M6. Therefore, the input-referred offset of OP05B is divided by the preamplifier gain and this resulting offset is added to the input-referred offset of the preamplifier. To limit the preamplifier offset and 1/f noise, large dimensions have to be used to keep the symmetry of the differential pair and to avoid mismatches between both inputs. A smaller area alternative would be to consider well-known architectures [Using a standard Miller Operational Amplifier, from the analog IP library of the technology (AMS CMOS 0.35 μm), as a comparator, the automatic procedure leads to a degradation of the maximum final offset after fine tuning. This is due to the amplifier offset that is much larger than the targeted offset. From the datasheet of the IP, this phenomenon is obvious as the offset of the operational amplifier is guaranteed between ±7 mV. To fix this, an additional differential gain stage is added as a preamplifier to a standard Miller Operational Amplifier, namely OP05B, as depicted in tectures  such as A set of 500 Monte Carlo simulations is then used to compare the OP05B IP with the so-obtained low-offset comparator in terms of input-referred offset. With a typical preamplifier gain of 25, an input-referred offset of ±300 μV is observed, thus resulting in a reduction of the offset by a factor of about 20. This performance level is sufficient for a proof of concept, even if further developments may concern the design of a specific low-offset comparator avoiding the use of a standard Miller operational amplifier and thus limiting silicon area and power consumption while improving residual offset.FR. Additionally, the minimum PSRR is increased up to 78 dB, as illustrated in Using the previously proposed comparator and an automatic procedure to implement a fine tuning of the residual offset, a maximum final offset of ±500 µV is obtained from a set of 1000 Monte Carlo simulations cf. a. This vFiR, of about 1 Ω. This value must be greater than the maximum possible mismatch between the on-resistance of two consecutive switches, iD and i+D1. This mismatch has been characterized using MC simulations and it appears that a worst-case ∆onR of 164 mΩ has been obtained for transistor with Last but not least, design must pay attention to monotonicity of linear potentiometers. This is particularly true for the fine-tuning potentiometer that involves very small elementary resistances, ddV, bridge nominal resistance, R0, and resistance variations, ΔR. Since ΔR/R0, for a given input magnitude, is generally set by fabrication, and is independent of R0, WB output voltage may be adjusted linearly with supply voltage. In general, depending on application, sensors have a scale factor requirement. Then, if the initial value of the scale factor is higher than this requirement, downscaling may be applied using two potentiometers, gndR and VddR, as depicted in According to Equation (1), output voltage of a given sensor depends on supply voltage, gndR and VddR are increased symmetrically from 0 to 930 Ω using five configuration bits and elementary steps of 30 Ω. An example is presented in ddV/2 but it is slightly decreased during coarse tuning of offset due to the added resistors between ddV and bV1, bV2 and to digital potentiometers A and B that are added in parallel with R2+ and R2− (gndR and VddR. According to (1), a sensitivity of 50 mV/% is obtained for a full bridge configuration and a supply voltage of 5 V. After offset compensation , the mean value of this sensitivity is reduced up to about 10% (45 mV/%) due to the presence of compensation resistances that are added in series and parallel with sensor resistors. To mimic the impact of process scattering on sensitivity, a gaussian distribution with a 10% maximal deviation of the so-obtained scale factor has been modeled. Consequently a σ of 3.33% of the mean value is observed for the scale factor. To reduce this dispersion, any value of the scale factor, lower than the minimum of the MC distribution, can be targeted. As an example, a sensitivity target of 40 mV/% is chosen. To reach this target, both values of  and R2− . It is wgndR and VddR. Results of Monte Carlo simulations are presented in Offset compensation range: ±30 mV,Power supply voltage: 5 V,R0 = 5 kΩ,Nominal resistance in the Wheatstone bridge: osR = 75 Ω with n = 15 and a rpolyh (1 kΩ/sq.) resistance 100osR = 500 Ω.For potentiometer C, design choice consisted in implementing a rpoly2 (50 Ω/sq.) resistance nAs a consequence, potentiometer A and B implements a resistance ranging from 41 kΩ up to 50 kΩ in n steps. To cancel-out effect of temperature coefficient of resistances, rpoly2 and rpolyh are also used accordingly to potentiometer C.A first demonstrator has been fabricated in a 0.35 μm Technology from AMS . For digMoreover, a seven-bits linear potentiometer with a maximum resistance of about 7.5 kΩ has been added to adjust sensitivity, and an internal FSM with 32 states has been used to control a linear potentiometer with 14 resistances of 1Ω .2 in silicon area. Most of the silicon is occupied by control I/Os that have been implemented as a parallel bus in this first demonstrator. Obviously, a future version of the smart-trimming IC will include an SPI control and on-chip non-volatile memories to reduce both footprint and numbers of wires to be bounded. A rough evaluation allows to estimate the size of the die that could be used to replace the laser trimming die to about 1 mm2 including I/Os in this technology.ASIC is less than 500 × 500 µmR1+, R4−, R2− and R3+—and the trimming network resistors depicted in red on the schematic with their initial values before laser-induced increase. After disconnecting the trimming network, we connected the proposed smart-trimming IC to the bare sensor, as illustrated on For the purpose of the proof of concept, we have used a commercial piezo-resistive pressure sensor that includes in a single package the sensor and a laser-trimming die. The so-obtained internal electrical schematic a is compAfter removal of the trimming die, the bare sensor offset has been measured at around −8 mV at ambient temperature. n = 30 down to ±1 mV after compensation. To verify the full range of operation of the CTO, we added a linear potentiometer in one branch of the Wheatstone bridge to emulate sensors with initial offset between −40 mV and −8 mV for code 00000. Then, putting the linear potentiometer in the other branch, initial offset between −8 mV and +40 mV are emulated. For each position of the potentiometer, the initial offset is measured with code 00000 and the residual offset after compensation with the best code is reported versus the initial offset b. ExperiThen, TDO has been characterized . For thet = 0, output voltage is equal to +700 µV due to mismatches between the four discrete resistors and the FSM starts decrementing output voltage by steps of about 150 μV till the output voltage sign changes. It can be noticed that the first step is twice the others due to a mismatch effect at the boundaries of the network of resistors. This effect could be reduced or even cancelled by a redesign of the layout and the addition of dummy structures at the boundaries of the network of resistors. The internal clock frequency is characterized from the width of each steps: the clock period is equal to about 4 ms corresponding to a clock frequency of about 260 Hz. On this specific example, the residual offset after fine tuning of offset is equal to −58 μV.As previously mentioned, automatic fine-tuning of offset cannot be validated on this first demonstrator. A specific experiment based on four identical resistors of 4.7 kΩ is then used to verify the fine-tuning steps, the internal clock frequency and the comparator offset. gndR and VddR on Finally, sensitivity adjustment has been verified. This electrical trimming is controlled by a seven bits digital signal to control potentiometer n2 using only two digital potentiometers with n discrete elements. Integration, after analysis and improvement, of a state-of-the-art solution for cancelation of thermal drift of offset has been also validated thus allowing to significantly improve the minimum detectable signal at the output of a WB. Scale Factor calibration has been also implemented independently to further improve specifications of a COTS sensor. It is worth noting that even if the architecture is generic, an ASIC design may not be optimal for any sensor. For example, if resistances of several tens or even hundreds of kΩ are implemented in the sensor, integrated potentiometers must be adapted to the post-calibration requested performance in terms of offset, PSRR and scale factor. Last but not least, ageing compensation and re-calibration is possible for high-end sensors. To conclude, our proposal is particularly adapted for sensors that must behave as passive ones with performances of carefully calibrated ones.Overall, experimental results obtained with this first demonstrator are in-line with our expectations. By implementing coarse and fine tuning of offset, offset can be reduced by a ratio of about 2gnd and ddV terminals for each branch). Once connected to the sensor die, potentially in a single package, the so-obtained smart-sensor still looks like a four-terminal Wheatstone bridge and it is then possible to adjust both output offset and sensitivity using digital control inputs accessible only during calibration. Additionally, an on-chip fine-tuning of offset may be automatically launched on power-up or upon external request depending on the application. Degraded modes are possible such as, for example, if the ground terminal is common to both branches, as in the presented experiment, the fine tuning of offset at each power-up or upon request is no longer available and, for a fully connected Wheatstone bridge with only four terminals, scale factor adjustment, using VddR and gndR, and partial offset compensation, using potentiometers A and B, remain available.In this paper, a fully electrical architecture for smart post-fabrication trimming of resistive sensors has been proposed. It allows to easily compensate the impact of process variations on a resistive sensor thanks to a compact IC. The proposed architecture is generic and may apply to any resistive sensors with six independent terminals (i.e., with independent Electrical simulations based on intensive Monte Carlo simulations have demonstrated the main advantages of the proposed solution: fully electrical operation, significant reduction of offset and PSRR, fine adjustment of sensitivity. Application to a COTS five-terminal sensor connected to an ASIC demonstrator, fabricated in a 0.35 μm Technology from AMS, has been demonstrated successfully, and thermal drift of the smart-sensor offset has been identified to remain identical to the bare sensor one.Perspectives of this work concern different aspects to increase genericity and performance. First, the coarse tuning of offset can be adapted to cancel-out the offset thermal drift of the bare sensor if the theoretical work presented in