Cytosine-rich Oligonucleotides Research Articles

IMab antibody binds single-stranded cytosine-rich sequences and unfolds DNA i-motifs.

i-Motifs (iMs) are non-canonical, four-stranded secondary structures formed by stacking of hemi-protonated CH+·C base pairs in cytosine-rich DNA sequences, predominantly at pH<7. The presence of iM structures in cells was a matter of debate until the recent development of iM-specific antibody, iMab, which was instrumental for several studies that suggested the existence of iMs in live cells and their putative biological roles. We assessed the interaction of iMab with cytosine-rich oligonucleotides by biolayer interferometry (BLI), pull-down assay and bulk-FRET experiments. Our results suggest that binding of iMab to DNA oligonucleotides is governed by the presence of runs of at least two consecutive cytosines and is generally increased in acidic conditions, irrespectively of the capacity of the sequence to adopt, or not, an iM structure. Moreover, the results of the bulk-FRET assay indicate that interaction with iMab results in unfolding of iM structures even in acidic conditions, similarly to what has been observed with hnRNP K, well-studied single-stranded DNA binding protein. Taken together, our results strongly suggest that iMab actually binds to blocks of 2-3 cytosines in single-stranded DNA, and call for more careful interpretation of results obtained with this antibody.

Cytosine-Rich Oligonucleotide and Electrochemically Reduced Graphene Oxide Nanocomposite for Ultrasensitive Electrochemical Ag+ Sensing.

Silver ions (Ag+) are crucial in various fields, but pose environmental and health risks at high concentrations. This study presents a straightforward approach for the ultra-trace detection of Ag+, utilizing a composite of a cytosine-rich oligonucleotide (CRO) and an electrochemically reduced graphene oxide (ERGO). Initially, ERGO was synthesized on a glassy carbon electrode (GCE) through the reduction of graphene oxide (GO) via cyclic voltammetry. A methylene blue-tagged CRO (MB-CRO) was then anchored to the ERGO surface through π-π interactions, resulting in the formation of an MB-CRO-modified ERGO electrode (MB-CRO/ERGO-GCE). The interaction with Ag+ ions induced the formation of silver-mediated C-Ag+-C coordination, prompting the MB-CRO to adopt a hairpin structure. This conformational change led to the desorption of the MB-CRO from the ERGO-GCE, causing a variation in the redox current of the methylene blue associated with the MB-CRO. Electrochemical assays revealed that the sensor exhibits extraordinary sensitivity to Ag+ ions, with a linear detection range from 1 femtomolar (fM) to 100 nanomolars (nM) and a detection limit of 0.83 fM. Moreover, the sensor demonstrated high selectivity for Ag+ ions and several other benefits, including stability, reproducibility, and straightforward fabrication and operational procedures. Additionally, real sample analyses were performed using the modified electrode to detect Ag+ in tap and pond water samples, yielding satisfactory recovery rates.

Cytosine-rich oligonucleotides incorporating a non-nucleotide loop: A further step towards the obtainment of physiologically stable i-motif DNA

i-Motifs, also known as i-tetraplexes, are secondary structures of DNA occurring in cytosine-rich oligonucleotides (CROs) that recall increasing interest in the scientific community for their relevance in various biological processes and DNA nanotechnology. This study reports the design of new structurally modified CROs, named Double-Ended-Linker-CROs (DEL-CROs), capable of forming stable i-motif structures. Here, two C-rich strands having sequences d(AC4A) and d(C6) have been attached, in a parallel fashion, to the two linker's edges by their 3′ or 5′ ends. The resulting DEL-CROs have been investigated for their capability to form i-motif structures by circular dichroism, poly-acrylamide gel electrophoresis, HPLC–size-exclusion chromatography, and NMR studies. This investigation established that DEL-CROs could form more stable i-motif structures than the corresponding unmodified CROs. In particular, the i-motif formed by DEL-5′-d(C6)2 resulted stable enough to be detected even at near physiological conditions (37 °C, pH 7.0). The results open the way to developing pH-switchable nanocarriers and aptamers based on suitably functionalized DEL-CROs.

A novel fluorescent platform of DNA-stabilized silver nanoclusters based on exonuclease III amplification-assisted detection of Salmonella Typhimurium

A novel fluorescent platform of DNA-stabilized silver nanoclusters (DNA-AgNCs) has been developed based on exonuclease III (Exo III) amplification-assisted for simple and sensitive detection of Salmonella Typhimurium (S. Typhimurium). The platform was designed by using magnetic beads, aptamer, its complementary DNA, hairpin probe (HP), Exo III, AgNO3, and NaBH4. The functionalized HP contained a cytosine-rich oligonucleotide loop (C-rich loop), which served as an effective template for the chemical reduction of Ag+ with NaBH4 to synthesize DNA-AgNCs. In the presence of S. Typhimurium, the C-rich loop was converted into an open form of ssDNA by the recycle digestion of Exo III, leading to a corresponding decrease in fluorescence intensity. Based on the fluorescence changes of the formed DNA-AgNCs, the sensitive detection of S. Typhimurium was achieved. Under the optimal conditions, a wide linear relationship was observed in the concentration of S. Typhimurium ranging from 4.6 × 102 to 4.6 × 107 cfu mL−1 with the limit of detection (LOD) being 82 cfu mL−1. The method showed good selectivity for detecting S. Typhimurium. In addition, the platform could be used for the detection of S. Typhimurium in milk samples. The LOD reached 6.6 × 102 cfu mL−1 with a good linear range, indicating that the method had excellent practicability in complex food samples.

I-motif structures in long cytosine-rich sequences found upstream of the promoter region of the SMARCA4 gene

Cytosine-rich oligonucleotides are capable of forming complex structures known as i-motif with increasingly studied biological properties. The study of sequences prone to form i-motifs located near the promoter region of genes may be difficult because these sequences not only contain repeats of cytosine tracts of disparate length but also these may be separated by loops of varied nature and length. In this work, the formation of intramolecular i-motif structures by a long sequence located upstream of the promoter region of the SMARCA4 gene has been demonstrated. Nuclear Magnetic Resonance, Circular Dichroism, Gel Electrophoresis, Size-Exclusion Chromatography, and multivariate analysis have been used. Not only the wild sequence (5‘-TC3T2GCTATC3TGTC2TGC2TCGC3T2G2TCATGA2C4-3’) has been studied but also several other truncated and mutated sequences. Despite the apparent complex sequence, the results showed that the wild sequence may form a relatively stable and homogeneous unimolecular i-motif structure, both in terms of pH or temperature. The model ligand TMPyP4 destabilizes the structure, whereas the presence of 20% (w/v) PEG200 stabilized it slightly. This finding opens the door to the study of the interaction of these kind of i-motif structures with stabilizing ligands or proteins.

Colorimetric detection of cysteine and homocysteine based on an oligonucleotide-stabilized Pd nanozyme

Pd-based peroxidase nanomimetic materials were templated using cytosine-rich oligonucleotides, and were employed in the colorimetric detection of biothiols including cysteine and homocysteine.

A feasible C-rich DNA electrochemical biosensor based on Fe3O4@3D-GO for sensitive and selective detection of Ag+

It is highly desirable and a popular topic in scientific research to develop a highly sensitive and selective detection and quantitative analysis method of determining trace silver ions (Ag+) in aqueous media since Ag+ ions are recognized as one of the most hazardous metal pollutants. Three-dimensional graphene oxide (3D-GO) was decorated with Fe3O4 to form nanocomposites of Fe3O4@3D-GO, which were further used as electrode materials in an electrochemical DNA sensor for detecting Ag+. Because of the chemical functionality and porous structure of the developed nanocomposites, single-stranded and cytosine-rich oligonucleotides could be anchored onto their surfaces or interior, followed by selective detection of Ag+ through formation of C–Ag+–C complex forms. The results of electrochemical measurement showed that the Fe3O4@3D-GO nanocomposites exhibits high sensitivity for detecting Ag+ with a detection limit of 2.0 pM within the range of 0.01–100 nM Ag+ (S/N = 3). The developed electrochemical biosensor based on Fe3O4@3D-GO exhibits highly selectivity, good repeatability, and stability for detecting Ag+ ions. The results of this work demonstrate that Fe3O4@3D-GO nanocomposites could be used as a promising tool to detect heavy metal ions in water or the environment.

Hairpin DNA probe with 5′-TCC/CCC-3′ overhangs for the creation of silver nanoclusters and miRNA assay

A facile strategy for the assay of target miRNA using fluorescent silver nanoclusters (AgNCs) has been described. Due to the preferable interaction between cytosine residues and Ag+, a short cytosine-rich oligonucleotide (ODN) with only six bases 5′-TCCCCC-3′ served as an efficient scaffold for the creation of the AgNCs. The AgNCs displayed a bright red emission when excited at 545nm. Such ODN base-stabilized AgNCs have been exploited for miRNA sensing. Overhangs of TCC at the 5′ end (5′-TCC) and CCC at the 3′ end (CCC-3′) (denoted as 5′-TCC/CCC-3′) appended to the hairpin ODN probe which also contains recognition sequences for target miRNA were included. Interestingly, the AgNCs/hairpin ODN probe showed similar spectral properties as that templated by 5′-TCCCCC-3′. The formation of the hairpin ODN probe/miRNA duplex separated the 5′-TCC/CCC-3′ overhangs, thus disturbing the optical property or structure of the AgNCs. As a result, fluorescence quenching of the AgNCs/hairpin ODN probe was obtained, which allows for facile determination of target miRNA. The proposed method is simple and cost-effective, holding great promise for clinical applications.

Fluorescent detection of silver(I) and cysteine using SYBR Green I and a silver(I)-specific oligonucleotide

We report on a novel method for the determination of silver ion (Ag+) and cysteine (Cys) by using the probe SYBR Green I (SGI) and an Ag+-specific cytosine-rich oligonucleotide (C-DNA). The fluorescence of SGI is very weak in the absence or presence of randomly coiled C-DNA. If, however, C-DNA interacts with Ag+ through the formation of cytosine-Ag+-cytosine (C-Ag+-C) base pairs, the randomly coiled C-DNA undergoes a structural changes to form a hairpin-like structure, thereby increasing the fluorescence of SGI. This fluorescence turn-on process allows the detection of Ag+ in the 10–600 nM concentration range, with a detection limit of 4.3 nM. Upon the reaction of Ag+ with Cys, Cys specifically removes Ag+ from the C-Ag+-C base pairs and destroys the hairpin-like structure. This, in turn, results in a decrease in fluorescence intensity. This fluorescence turn-off process enables the determination of Cys in the 8–550 nM concentration range, with a detection limit of 4.5 nM. The method reported here for the determination of either Ag+ or Cys is simple, sensitive, and affordable, and may be applied to other detection systems if appropriately selected DNA sequences are available.

Primordial soup or vinaigrette: did the RNA world evolve at acidic pH?

BackgroundThe RNA world concept has wide, though certainly not unanimous, support within the origin-of-life scientific community. One view is that life may have emerged as early as the Hadean Eon 4.3-3.8 billion years ago with an atmosphere of high CO2 producing an acidic ocean of the order of pH 3.5-6. Compatible with this scenario is the intriguing proposal that life arose within alkaline (pH 9-11) deep-sea hydrothermal vents like those of the 'Lost City', with the interface with the acidic ocean creating a proton gradient sufficient to drive the first metabolism. However, RNA is most stable at pH 4-5 and is unstable at alkaline pH, raising the possibility that RNA may have first arisen in the acidic ocean itself (possibly near an acidic hydrothermal vent), acidic volcanic lake or comet pond. As the Hadean Eon progressed, the ocean pH is inferred to have gradually risen to near neutral as atmospheric CO2 levels decreased.Presentation of the hypothesisWe propose that RNA is well suited for a world evolving at acidic pH. This is supported by the enhanced stability at acidic pH of not only the RNA phosphodiester bond but also of the aminoacyl-(t)RNA and peptide bonds. Examples of in vitro-selected ribozymes with activities at acid pH have recently been documented. The subsequent transition to a DNA genome could have been partly driven by the gradual rise in ocean pH, since DNA has greater stability than RNA at alkaline pH, but not at acidic pH.Testing the hypothesisWe have proposed mechanisms for two key RNA world activities that are compatible with an acidic milieu: (i) non-enzymatic RNA replication of a hemi-protonated cytosine-rich oligonucleotide, and (ii) specific aminoacylation of tRNA/hairpins through triple helix interactions between the helical aminoacyl stem and a single-stranded aminoacylating ribozyme.Implications of the hypothesisOur hypothesis casts doubt on the hypothesis that RNA evolved in the vicinity of alkaline hydrothermal vents. The ability of RNA to form protonated base pairs and triples at acidic pH suggests that standard base pairing may not have been a dominant requirement of the early RNA world.ReviewersThis article was reviewed by Eugene Koonin, Anthony Poole and Charles Carter (nominated by David Ardell).

A graphene-based fluorescent nanoprobe for silver(i) ions detection by using graphene oxide and a silver-specific oligonucleotide

A fluorescence sensor for Ag(I) ions is developed based on the target-induced conformational change of a silver-specific cytosine-rich oligonucleotide (SSO) and the interactions between the fluorogenic SSO probe and graphene oxide.

Structural Polymorphism of Telomeric DNA Regulated by pH and Divalent Cation

DNA oligonucleotides can form multi-stranded structures such as a duplex, triplex, and quadruplex, while the double helical structure is generally considered as the canonical structure of DNA oligonucleotides. Guanine-rich or cytosine-rich oligonucleotides, which are observed in telomere, centromere, and other biologically important sequences in vivo, can form four-stranded G-quadruplex and I-motif structures in vitro. In this study, we have investigated the effects of pH and cation on the structures and their stabilities of d(G4T4G4) and d(C4A4C4). The CD spectra and thermal melting curves of DNAs at various pHs demonstrated that acidic conditions induced a stable I-motif structure of d(C4A4C4), while the pH value did not affect the G-quadruplex structure and stability of d(G4T4G4). The CD spectra of the 1 : 1 mixture of d(G4T4G4) and d(C4A4C4) indicated that the acidic conditions inhibit the duplex formation between d(G4T4G4) and d(C4A4C4). Isothermal titration calorimetry measurements of the duplex formation at various pHs also quantitatively indicated that the acidic conditions inhibit the duplex formation. On the other hand, the CD spectra and thermal melting curves of DNAs in the absence and presence of Ca2+ indicated that Ca2+ induces a parallel G-quadruplex sructure of d(G4T4G4) and then inhibits the duplex formation. These results lead to the conclusion that both the pH and coexisting cation can induce and regulate the structural polymorphisms the oligonucleotides in which they form the G-quadruplex, I-motif, and duplex depending on the conditions. Thus, the results reported here indicate pivotal roles of pH and coexisting cations in biological processes by regulating the conformational switching between the duplex and quadruplexes structures of the guanine-rich or cytosine-rich oligonucleotides in vivo.

Kinetic Analysis of the Cleavage of the Ribose Phosphodiester Bond within Guanine and Cytosine-Rich Oligonucleotides and Dinucleotides at 65–200 °C and Its Implications Concerning the Chemical Evolution of RNA

Abstract A monitoring method of rapid hydrothermal reactions was successfully applied to a kinetic analysis of the cleavage of a ribose phosphodiester bond within oligonucleotides and dinucleotides at 150–200 °C. The apparent rate constants (kapp) of degradation of the ribose 3′,5′-cytidylylguanosine sequence (-C3′pGd-) within oligonucleotides and dinucleotides were determined, where the -C3′pGd- sequence in oligonucleotides is less stable than 2′,5′-cytidylylguanosine (C2′pG) and 3′,5′-cytidylylguanosine (C3′pG). It was unexpected that the stability of the target sequence would be dependent on the surrounding sequences of the oligonucleotides, although the temperatures used in the study were extremely higher than the melting points. The stability of a phosphodiester bond of 2′-deoxycytidylyl-2′-deoxyguanosine (CdpGd) is much higher than that of a ribose phosphodiester bond at low temperatures, but becomes comparable at 200 °C. During the degradation of C2′pG or C3′pG, interconversion between C2′pG and C3′pG was observed along with cleavage of the phosphodiester bond. Based on an analysis of the extent of interconversion, the apparent rate constants of the disappearance of C2′pG and C3′pG were dissected into the rate constants of hydrolysis (khy) and interconversion (kint), where the values of khy were greater than those of kint. The apparent activation energy of the degradation of the target sequence was 100–109 kJ mol−1 for oligonucleotides, 90 kJ mol−1 for C3′pG, and 87 kJ mol−1 for C2′pG, and 139 kJ mol−1 for CdpGd. The apparent activation enthalpy and entropy changes of the degradation of the target sequence were also determined; the values of the activation parameter were ΔH≠app = 94–105 kJ mol−1 and ΔS≠app = −(36–59) J mol−1 T−1 for five oligonucleotides, ΔH≠app = 86 kJ mol−1 and ΔS≠app = −97 J mol−1 T−1 for C3′pG, ΔH≠app = 84 kJ mol−1, ΔS≠app = −105 J mol−1 T−1 for C2′pG, and ΔH≠app = 135 kJ mol−1, ΔS≠app = +2 J mol−1 T−1 for CdpGd. The activation parameters, ΔH≠app and ΔS≠app, for the oligonucleotides increased with the length of the surrounding sequence of -C3′pGd-; this fact clearly demonstrates the existence of the influence of the surrounding sequence for the stability of the target ribose phosphodiester bond. Based on a kinetic analysis, the reaction mechanism of the degradation of the ribose phosphodiester bond at high temperatures is discussed. Furthermore, possible pathways of the chemical evolution of RNA are discussed from the viewpoint of the hydrothermal origin of life.