Important Chemical Modifications Research Articles

Exploring the Benzazoles Derivatives as Pharmacophores for AChE, BACE1, and as Anti-Aβ Aggregation to Find Multitarget Compounds against Alzheimer’s Disease

Despite the great effort that has gone into developing new molecules as multitarget compounds to treat Alzheimer’s disease (AD), none of these have been approved to treat this disease. Therefore, it will be interesting to determine whether benzazoles such as benzimidazole, benzoxazole, and benzothiazole, employed as pharmacophores, could act as multitarget drugs. AD is a multifactorial disease in which several pharmacological targets have been identified—some are involved with amyloid beta (Aβ) production, such as beta secretase (BACE1) and beta amyloid aggregation, while others are involved with the cholinergic system as acetylcholinesterase (AChE) and butirylcholinesterase (BChE) and nicotinic and muscarinic receptors, as well as the hyperphosphorylation of microtubule-associated protein (tau). In this review, we describe the in silico and in vitro evaluation of benzazoles on three important targets in AD: AChE, BACE1, and Aβ. Benzothiazoles and benzimidazoles could be the best benzazoles to act as multitarget drugs for AD because they have been widely evaluated as AChE inhibitors, forming π–π interactions with W286, W86, Y72, and F338, as well as in the AChE gorge and catalytic site. In addition, the sulfur atom from benzothiazol interacts with S286 and the aromatic ring from W84, with these compounds having an IC50 value in the μM range. Also, benzimidazoles and benzothiazoles can inhibit Aβ aggregation. However, even though benzazoles have not been widely evaluated on BACE1, benzimidazoles evaluated in vitro showed an IC50 value in the nM range. Therefore, important chemical modifications could be considered to improve multitarget benzazoles’ activity, such as substitutions in the aromatic ring with electron withdrawal at position five, or a linker 3 or 4 carbons in length, which would allow for better interaction with targets.

Syntheses, Properties, and Applications of Aminated‐Starch: A Review

AbstractBecause aminated‐starch (AS) is an important chemical modification strategy of starch, much research on AS has been carried out. The present review summarizes the main synthetic strategies for AS. This review also outlines the changes in starch properties due to amination. The effects of amination on particle morphology, crystallinity, thermal properties, adhesion, and cytotoxicity are specifically reviewed. The main applications of AS are also detailed, including adsorbent and nano‐loaded particles.

Fat mass and obesity-associated protein (FTO) mediated m6A modification of circFAM192A promoted gastric cancer proliferation by suppressing SLC7A5 decay

Gastric cancer (GC) is a common malignant tumor worldwide, especially in East Asia, with high incidence and mortality rate. Epigenetic modifications have been reported to participate in the progression of gastric cancer, among which m6A is the most abundant and important chemical modification in RNAs. Fat mass and obesity-associated protein (FTO) is the first identified RNA demethylase but little is known about its role in gastric cancer. In our study, data from TCGA and clinical samples showed that FTO was highly expressed in gastric cancer tissues. Kaplan–Meier plotter suggested that patients with the high level of FTO had a poor prognosis. In vitro and in vivo experiments confirmed the role of FTO in promoting gastric cancer cell proliferation. Mechanistically, we found that FTO bound to circFAM192A at the specific site and removed the m6A modification in circFAM192A, protecting it from degradation. CircFAM192A subsequently interacted with the leucine transporter solute carrier family 7 member 5 (SLC7A5) and enhancing its stability. As a result, an increased amount of SLC7A5 was on the membrane, which facilitated leucine uptake and activated the mTOR signaling pathway. Therefore, our study demonstrated that FTO promoted gastric cancer proliferation through the circFAM192A/SLC7A5 axis in the m6A-dependent manner. Our study shed new light on the role of FTO in gastric cancer progression.

NSUN2 promotes osteosarcoma progression by enhancing the stability of FABP5 mRNA via m5C methylation

5-methylcytosine (m5C) modification, which is mainly induced by the RNA methyltransferase NSUN2 (NOP2/Sun domain family, member 2), is an important chemical posttranscriptional modification in mRNA and has been proven to play important roles in the progression of many cancers. However, the functions and underlying molecular mechanisms of NSUN2-mediated m5C in osteosarcoma (OS) remain unclear. In this study, we found NSUN2 was highly expressed in OS tissues and cells. We also discovered that higher expression of NSUN2 predicted poorer prognosis of OS patients. Our study showed that NSUN2 could promote the progression of OS cells. Moreover, we employed RNA sequencing, RNA immunoprecipitation (RIP), and methylated RIP to screen and validate the candidate targets of NSUN2 and identified FABP5 as the target. We observed that NSUN2 stabilized FABP5 mRNA by inducing m5C modification and further promoted fatty acid metabolism in OS cells. Moreover, both knocking down the expression of FABP5 and adding fatty acid oxidation inhibitor could counterbalance the promoting effect of NSUN2 on the progression of OS. Our study confirms that NSUN2 can up-regulate the expression of FABP5 by improving the stability of FABP5 mRNA via m5C, so as to promote fatty acid metabolism in OS cells, and finally plays the role in promoting the progression of OS. Our findings suggest that NSUN2 is a promising prognostic marker for OS patients and may serve as a potential therapeutic target for OS treatment.A schematic illustration was proposed to summarize our findings.

4mCBERT: A computing tool for the identification of DNA N4-methylcytosine sites by sequence- and chemical-derived information based on ensemble learning strategies

N4-methylcytosine (4mC) is an important DNA chemical modification pattern which is a new methylation modification discovered in recent years and plays critical roles in gene expression regulation, defense against invading genetic elements, genomic imprinting, and so on. Identifying 4mC site from DNA sequence segment contributes to discovering more novel modification patterns. In this paper, we present a model called 4mCBERT that encodes DNA sequence segments by sequence characteristics including one-hot, electron-ion interaction pseudopotential, nucleotide chemical property, word2vec and chemical information containing physicochemical properties (PCP), chemical bidirectional encoder representations from transformers (chemical BERT) and employs ensemble learning framework to develop a prediction model. PCP and chemical BERT features are firstly constructed and applied to predict 4mC sites and show positive contributions to identifying 4mC. For the Matthew's Correlation Coefficient, 4mCBERT significantly outperformed other state-of-the-art models on six independent benchmark datasets including A. thaliana, C. elegans, D. melanogaster, E. coli, G. Pickering, and G. subterraneous by 4.32 % to 24.39 %, 2.52 % to 31.65 %, 2 % to 16.49 %, 6.63 % to 35.15, 8.59 % to 61.85 %, and 8.45 % to 34.45 %. Moreover, 4mCBERT is designed to allow users to predict 4mC sites and retrain 4mC prediction models. In brief, 4mCBERT shows higher performance on six benchmark datasets by incorporating sequence- and chemical-driven information and is available at http://cczubio.top/4mCBERT and https://github.com/abcair/4mCBERT.

Recognition of G-quadruplex RNA by a crucial RNA methyltransferase component, METTL14.

N6-methyladenosine (m6A) is an important epitranscriptomic chemical modification that is mainly catalyzed by the METTL3/METTL14 RNA methyltransferase heterodimer. Although m6A is found at the consensus sequence of 5′-DRACH-3′ in various transcripts, the mechanism by which METTL3/METTL14 determines its target is unclear. This study aimed to clarify the RNA binding property of METTL3/METTL14. We found that the methyltransferase heterodimer itself has a binding preference for RNA G-quadruplex (rG4) structures, which are non-canonical four-stranded structures formed by G-rich sequences, via the METTL14 RGG repeats. Additionally, the methyltransferase heterodimer selectively methylated adenosines close to the rG4 sequences. These results suggest a possible process for direct recruitment of METTL3/METTL14 to specific methylation sites, especially near the G4-forming regions. This study is the first to report the RNA binding preference of the m6A writer complex for the rG4 structure and provides insights into the role of rG4 in epitranscriptomic regulation.

Phosphorothioate Substitutions in RNA Structure Studied by Molecular Dynamics Simulations, QM/MM Calculations, and NMR Experiments.

Phosphorothioates (PTs) are important chemical modifications of the RNA backbone where a single nonbridging oxygen of the phosphate is replaced with a sulfur atom. PT can stabilize RNAs by protecting them from hydrolysis and is commonly used as a tool to explore their function. It is, however, unclear what basic physical effects PT has on RNA stability and electronic structure. Here, we present molecular dynamics (MD) simulations, quantum mechanical (QM) calculations, and NMR spectroscopy measurements, exploring the effects of PT modifications in the structural context of the neomycin-sensing riboswitch (NSR). The NSR is the smallest biologically functional riboswitch with a well-defined structure stabilized by a U-turn motif. Three of the signature interactions of the U-turn: an H-bond, an anion-π interaction, and a potassium binding site; are formed by RNA phosphates, making the NSR an ideal model for studying how PT affects RNA structure and dynamics. By comparing with high-level QM calculations, we reveal the distinct physical properties of the individual interactions facilitated by the PT. The sulfur substitution, besides weakening the direct H-bond interaction, reduces the directionality of H-bonding while increasing its dispersion and induction components. It also reduces the induction and increases the dispersion component of the anion-π stacking. The sulfur force-field parameters commonly employed in the literature do not reflect these distinctions, leading to the unsatisfactory description of PT in simulations of the NSR. We show that it is not possible to accurately describe the PT interactions using one universal set of van der Waals sulfur parameters and provide suggestions for improving the force-field performance.

Cellular uptake of polymeric nanoparticles by bovine cumulus-oocyte complexes and their effect on in vitro developmental competence

Polymeric nanoparticles (NPs) are produced using bio-compatible and bio-degradable materials such as PLGA (Poly(lactic-co-glycolic acid)). This technology provides a valuable tool to deliver molecules to the subcellular level with a relatively low risk of cytotoxicity. However their use in the field of reproductive biotechnology is not yet scientifically substantiated. The aim of the present study was to test if PLGA NPs can be taken-up by cumulus-enclosed oocytes as a first step towards potential oocyte-targeted applications to enhance oocyte quality and fertility. We conducted a series of experiments using bovine in vitro oocyte maturation as a model to study FITC-conjugated PLGA internalization (using laser-scanning confocal microscopy) and the effect of some important physical (particle size) and chemical (conjugation with PEG) modifications. We show evidence that PLGA NPs can be taken-up by cumulus cells and to a less extent by the enclosed oocytes regardless of the NP size. The NP transfer to the oocyte appear to be transcellular (via cumulus cells and transzonal projections) and paracellular (via zona pellucida). The PLGA NPs were detected in the vicinity of the oocyte as quick as 2 h post-exposure in a protein-free medium and did not compromise cumulus cell viability nor subsequent early embryo development or embryo quality. These results suggest that PLGA NPs may have promising applications as carriers for drug or molecule delivery targeting cumulus cells and oocytes.

Detection and Application of 5-Formylcytosine and 5-Formyluracil in DNA.

Nucleic acids contain a variety of different base modifications, such as decoration at the fifth position of cytosine, which is one of the most important epigenetic modifications. Nucleic acid epigenetics mediate a wide variety of biological processes, including embryonic development and gene regulation, genomic imprinting, differentiation, and X-chromosome inactivation. Furthermore, the modification level can be aberrantly expressed in distinct sets of tissue that can indicate different tumor onsets and canceration. Thus, the analysis of modified nucleobases may contribute to the understanding of epigenetic modification-related biological processes and the correlation of modified nucleobase patterns with disease states for clinical diagnosis and treatment. In addition to 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine are found in organisms at a low content but are nevertheless extremely important chemical modifications, and 5-hydroxyuracil and 5-formyluracil compounds are also present. 5-Formyluracil is found in bacteriophages, prokaryotes, and mammalian cells. The 5-formyluracil content is higher in certain cancer tissues than in the normal tissues adjacent to the tumor. The content of 5-formyluracil in different cell tissues may have cell type specificity. With the continuous use of chemical tools, new detection technologies have greatly advanced the research on natural pyrimidine modifications. These modifications dynamically regulate the gene expression in eukaryotes and prokaryotes and provide mechanistic insights into the occurrence of diseases. Natural pyrimidine modifications act not only as intermediates for DNA demethylation or oxidative damage products but also as modulators of gene expression. Therefore, the development of more effective chemical tools will help us better understand the dynamic changes of natural pyrimidine modifications in vivo. In this Account, we summarize the recent advanced techniques for the detection of 5-formylpyrimidine (5-formylcytosine and 5-formyluracil) and highlight their great potential as biomarkers in biomedical applications. Focusing on the great urgency for the detection of epigenetic modifications, our group developed a series of methods for the qualitative and quantitative analysis of 5-formylpyrimidine in the past few years, aiming at facilitating the accurate detection and mapping of these epigenetic modifications. By the construction of probes, 5-formylpyrimidine can be selectively labeled. Using mass spectrometry, the epigenetic modifications can be quantified. Upon treatment under specific conditions, 5-formylcytosine can be recognized at single-base resolution. With this Account, we anticipate providing chemical and biological researchers with some insight to unlock the complex mechanism involved in 5-formylpyrimidine-related biological processes and stimulate more collaborative research interests from the different fields of materials, biological, medicine, and chemistry to promote the translational research of epigenetics in tumor diagnosis and treatment.

KSIMC: Predicting Kinase⁻Substrate Interactions Based on Matrix Completion.

Protein phosphorylation is an important chemical modification catalyzed by kinases. It plays important roles in many cellular processes. Predicting kinase–substrate interactions is vital to understanding the mechanism of many diseases. Many computational methods have been proposed to identify kinase–substrate interactions. However, the prediction accuracy still needs to be improved. Therefore, it is necessary to develop an efficient computational method to predict kinase–substrate interactions. In this paper, we propose a novel computational approach, KSIMC, to identify kinase–substrate interactions based on matrix completion. Firstly, the kinase similarity and substrate similarity are calculated by aligning sequence of kinase–kinase and substrate–substrate, respectively. Then, the original association network is adjusted based on the similarities. Finally, the matrix completion is used to predict potential kinase–substrate interactions. The experiment results show that our method outperforms other state-of-the-art algorithms in performance. Furthermore, the relevant databases and scientific literature verify the effectiveness of our algorithm for new kinase–substrate interaction identification.

Correlation of electrical characteristics with interface chemistry and structure in Pt/Ru/PbZr0.52Ti0.48O3/Pt capacitors after post metallization annealing

We report the effect of post-metallization annealing (PMA) on the electrical behavior of Pt/Ru/PbZr0.52Ti0.48O3(PZT)/Pt capacitors and correlations with the physical chemistry of the top electrode/PZT interface. PMA improves the electrical characteristics, in particular the breakdown field while inducing important chemical and structural modifications at the interface. The Ru electrode layer is oxidized and disrupted. There is evidence for the formation of RuOx and ZrRuOx metallic phases at the interface but no Pb transport into the electrode region is observed.

Developments in Hydroxypropylation of Starch: A Review

Because hydroxypropyl starch (HPS) represents an important chemical modification of starch, extensive studies on hydroxypropylation are being carried out at various research centers worldwide. The present review summarizes development of methods for determination of the MS (molar substitution)/DS (degree of substitution) of hydroxypropyl starch, location of hydroxypropylation reaction sites in granules and on amylose and amylopectin molecules, and factors affecting reaction efficiency. Also presented is an overview of changes of physicochemical properties of starch that result from hydroxypropylation. Specifically reviewed are the effects of hydroxypropylation on granule morphology, crystallinity, thermal properties, swelling, and solubility; pasting, paste, and retrogradation properties; rheological and textural properties of HPS gels; and digestibility and uses of HPS.

A bionic system with Fenton reaction and bacteria as a model for bioprocessing lignocellulosic biomass

BackgroundThe recalcitrance of lignocellulosic biomass offers a series of challenges for biochemical processing into biofuels and bio-products. For the first time, we address these challenges with a biomimetic system via a mild yet rapid Fenton reaction and lignocellulose-degrading bacterial strain Cupriavidus basilensis B-8 (here after B-8) to pretreat the rice straw (RS) by mimicking the natural fungal invasion process. Here, we also elaborated the mechanism through conducting a systematic study of physicochemical changes before and after pretreatment.ResultsAfter synergistic Fenton and B-8 pretreatment, the reducing sugar yield was increased by 15.6–56.6% over Fenton pretreatment alone and 2.7–5.2 times over untreated RS (98 mg g−1). Morphological analysis revealed that pretreatment changed the surface morphology of the RS, and the increase in roughness and hydrophilic sites enhanced lignocellulose bioavailability. Chemical components analyses showed that B-8 removed part of the lignin and hemicellulose which caused the cellulose content to increase. In addition, the important chemical modifications also occurred in lignin, 2D NMR analysis of the lignin in residues indicated that the Fenton pretreatment caused partial depolymerization of lignin mainly by cleaving the β-O-4 linkages and by demethoxylation to remove the syringyl (S) and guaiacyl (G) units. B-8 could depolymerize amount of the G units by cleaving the β-5 linkages that interconnect the lignin subunits.ConclusionsA biomimetic system with a biochemical Fenton reaction and lignocellulose-degrading bacteria was confirmed to be able for the pretreatment of RS to enhance enzymatic hydrolysis under mild conditions. The high digestibility was attributed to the destruction of the lignin structure, partial hydrolysis of the hemicellulose and partial surface oxidation of the cellulose. The mechanism of synergistic Fenton and B-8 pretreatment was also explored to understand the change in the RS and the bacterial effects on enzymatic hydrolysis. Furthermore, this biomimetic system offers new insights into the pretreatment of lignocellulosic biomass.

New Insights in the Design of Bioactive Peptides and Chelating Agents for Imaging and Therapy in Oncology.

Many synthetic peptides have been developed for diagnosis and therapy of human cancers based on their ability to target specific receptors on cancer cell surface or to penetrate the cell membrane. Chemical modifications of amino acid chains have significantly improved the biological activity, the stability and efficacy of peptide analogues currently employed as anticancer drugs or as molecular imaging tracers. The stability of somatostatin, integrins and bombesin analogues in the human body have been significantly increased by cyclization and/or insertion of non-natural amino acids in the peptide sequences. Moreover, the overall pharmacokinetic properties of such analogues and others (including cholecystokinin, vasoactive intestinal peptide and neurotensin analogues) have been improved by PEGylation and glycosylation. Furthermore, conjugation of those peptide analogues to new linkers and bifunctional chelators (such as AAZTA, TETA, TRAP, NOPO etc.), produced radiolabeled moieties with increased half life and higher binding affinity to the cognate receptors. This review describes the most important and recent chemical modifications introduced in the amino acid sequences as well as linkers and new bifunctional chelators which have significantly improved the specificity and sensitivity of peptides used in oncologic diagnosis and therapy.

Understanding the structural and chemical changes of plant biomass following steam explosion pretreatment

BackgroundBiorefining of lignocellulosic biomass has become one of the most valuable alternatives for the production of multi-products such as biofuels. Pretreatment is a prerequisite to increase the enzymatic conversion of the recalcitrant lignocellulose. However, there is still considerable debate regarding the key features of biomass impacting the cellulase accessibility. In this study, we evaluate the structural and chemical features of three different representative biomasses (Miscanthus × giganteus, poplar and wheat straw), before and after steam explosion pretreatment at increasing severities, by monitoring chemical analysis, SEM, FTIR and 2D NMR.ResultsRegardless the biomass type, combined steam explosion pretreatment with dilute sulfuric acid impregnation resulted in significant improvement of the cellulose conversion. Chemical analyses revealed that the pretreatment selectively degraded the hemicellulosic fraction and associated cross-linking ferulic acids. As a result, the pretreated residues contained mostly cellulosic glucose and lignin. In addition, the pretreatment directly affected the cellulose crystallinity but these variations were dependent upon the biomass type. Important chemical modifications also occurred in lignin since the β-O-4′ aryl-ether linkages were found to be homolytically cleaved, followed by some recoupling/recondensation to β-β′ and β-5′ linkages, regardless the biomass type. Finally, 2D NMR analysis of the whole biomass showed that the pretreatment preferentially degraded the syringyl-type lignin fractions in miscanthus and wheat straw while it was not affected in the pretreated poplar samples.ConclusionsOur findings provide an enhanced understanding of parameters impacting biomass recalcitrance, which can be easily generalized to both woody and non-woody biomass species. Results indeed suggest that the hemicellulose removal accompanied by the significant reduction in the cross-linking phenolic acids and the redistribution of lignin are strongly correlated with the enzymatic saccharification, by loosening the cell wall structure thus allowing easier cellulase accessibility. By contrast, we have shown that the changes in the syringyl/guaiacyl ratio and the cellulose crystallinity do not seem to be relevant factors in assessing the enzymatic digestibility. Some biomass type-dependent and easily measurable FTIR factors are highly correlated to saccharification.

Site-selective chemical cleavage of peptide bonds.

Site-selective cleavage of extremely unreactive peptide bonds is a very important chemical modification that provides invaluable information regarding protein sequence, and it acts as a modulator of protein structure and function for therapeutic applications. For controlled and selective cleavage, a daunting task, chemical reagents must selectively recognize or bind to one or more amino acid residues in the peptide chain and selectively cleave a peptide bond. Building on this principle, we have developed an approach that utilizes a chemical reagent to selectively modify the serine residue in a peptide chain and leads to the cleavage of a peptide backbone at the N-terminus of the serine residue. After cleavage, modified residues can be converted back to the original fragments. This method exhibits broad substrate scope and selectively cleaves various bioactive peptides with post-translational modifications (e.g. N-acetylation and -methylation) and mutations (d- and β-amino acids), which are a known cause of age related diseases.

Long-term trends of chemical and modelled photochemical parameters in four Alpine lakes

Based on long-term trends of water chemistry parameters of photochemical significance from four lakes located in the Alps (Iseo, Garda, Piburgersee, Geneva), we calculated the corresponding steady-state concentrations of photoinduced transient species with an ad-hoc photochemical model. Such transients were the hydroxyl (•OH) and carbonate (CO3−•) radicals, singlet oxygen (1O2), and the triplet states of chromophoric dissolved organic matter (3CDOM*). Among the investigated lakes, Lake Iseo, for example, showed a long-term near-stability in chemical parameters that resulted in a photochemical stability. By contrast, Piburgersee underwent important chemical modifications, but the interplay of compensation (parallel increase of both inorganic and organic carbon) and near-saturation effects (organic matter as main •OH source and sink) prevented the modelled photochemistry to undergo significant shifts over time. This result suggests the occurrence of a sort of “photochemical buffering” in some lake ecosystems, which would dampen modifications of the steady-state concentration of the photochemically-formed reactive transients, even in the case of significant changes in water chemistry. Finally, in lakes Garda and Geneva, long-term changes in water chemistry had an effect on photochemistry. While in Lake Garda the small increase in DOM was associated to a small increase in 1O2 and 3CDOM*, in Lake Geneva, the increases in pH and bicarbonate and the decrease in nitrite resulted in an •OH decrease. Overall, our results predict very different lake photochemistry patterns in relation to alterations in water chemistry parameters caused by climate change, such as changes in water alkalinity and dissolved organic carbon concentration.

Surface Analysis and Ultra‐Shallow Molecular Depth‐Profiling of Polyethylene Treated by an Atmospheric Ar‐D2O Post‐Discharge

In an original approach to trace water reactivity upon surface treatment by ambient “open air” plasma, the H–D exchange processes induced in polyethylene by an Ar‐D2O post‐discharge are investigated using time‐of‐flight secondary ion mass spectrometry, both at the surface and along the sample depth. The surface characterization points out the strong influence of the sample‐torch distance. The most important chemical modifications, excluding polymer overheating, occur at 5 mm of distance, with 25% of deuterated monomer units at the surface. For the first time, ultra‐shallow molecular depth‐profiling by Ar noble gas clusters shows the detailed variation of the deuteration of the polymer repeat unit in the topmost surface layer. Our results indicate that the fraction of deuterated monomer units is reduced by a factor >2 over a depth of ∼3 nm.

Effect of Acid Hydrolysis on Starch Structure and Functionality: A Review

Acid hydrolysis is an important chemical modification that can significantly change the structural and functional properties of starch without disrupting its granular morphology.A deep understanding of the effect of acid hydrolysis on starch structure and functionality is of great importance for starch scientific research and its industrial applications. During acid hydrolysis, amorphous regions are hydrolyzed preferentially, which enhances the crystallinity and double helical content of acid hydrolyzed starch. This review discusses current understanding of the effect of acid hydrolysis on starch structure and functionality. The effects of acid hydrolysis on amylose content, chain length distribution of amylopectin molecules, molecular and crystalline organization (including lamellar structure) and granular morphology are considered. Functional properties discussed include swelling power, gelatinization, retrogradation, pasting, gel texture, and in vitro enzyme digestibility. The paper also highlights some promising applications of acid hydrolyzed starch (starch nanocrystals) in the preparation of biodegradable nanocomposites, bio-hydrogen, and slowly digestible starch-based healthy foods.

Cy3 and Cy5 dyes attached to oligonucleotide terminus stabilize DNA duplexes: Predictive thermodynamic model

Cyanine dyes are important chemical modifications of oligonucleotides exhibiting intensive and stable fluorescence at visible light wavelengths. When Cy3 or Cy5 dye is attached to 5′ end of a DNA duplex, the dye stacks on the terminal base pair and stabilizes the duplex. Using optical melting experiments, we have determined thermodynamic parameters that can predict the effects of the dyes on duplex stability quantitatively (ΔG°, Tm). Both Cy dyes enhance duplex formation by 1.2kcal/mol on average, however, this Gibbs energy contribution is sequence-dependent. If the Cy5 is attached to a pyrimidine nucleotide of pyrimidine–purine base pair, the stabilization is larger compared to the attachment to a purine nucleotide. This is likely due to increased stacking interactions of the dye to the purine of the complementary strand. Dangling (unpaired) nucleotides at duplex terminus are also known to enhance duplex stability. Stabilization originated from the Cy dyes is significantly larger than the stabilization due to the presence of dangling nucleotides. If both the dangling base and Cy3 are present, their thermodynamic contributions are approximately additive. New thermodynamic parameters improve predictions of duplex folding, which will help design oligonucleotide sequences for biophysical, biological, engineering, and nanotechnology applications.