Duplex Stability Research Articles

A Quencher-Free Linear Probe from Serinol Nucleic Acid with a Fluorescent Uracil Analogue.

With the goal of developing a quencher-free probe composed of an artificial nucleic acid, the fluorescent nucleobase analogue 5-(perylenylethynyl)uracil (Pe U), which was incorporated into totally artificial serinol nucleic acid (SNA) as a substitute for thymine, has been synthesized. In the context of a 12-mer duplex with RNA, these fluorophores reduce duplex stability slightly compared with that of an SNA without Pe U modification; thus suggesting that structural distortion is not induced by the modification. If two Pe Us were incorporated at separate positions in an SNA, the fluorescent emission at λ≈490 nm was clearly enhanced upon hybridization with complementary RNA. A quencher-free SNA linear probe containing three Pe Us, each separated by six nucleobases, has been designed. Detection of target RNA with high sensitivity and discrimination of a single-base mismatch has also been demonstrated.

Silver(I)-Ion-Mediated Cytosine-Containing Base Pairs: Metal Ion Specificity for Duplex Stabilization and Susceptibility toward DNA Polymerases.

Spectroscopic characterization of AgI -ion-mediated C-AgI -A and C-AgI -T base pairs found in primer extension reactions catalyzed by DNA polymerases was conducted. UV melting experiments revealed that C-A and C-T mismatched base pairs in oligodeoxynucleotide duplexes are specifically stabilized by AgI ions in 1:1 stoichiometry in the same manner as a C-C mismatched base pair. Although the stability of the mismatched base pairs in the absence of AgI ions is in the order C-A≈C-T>C-C, the stabilizing effect of AgI ions follows the order C-C>C-A≈C-T. However, the comparative susceptibility of dNTPs to AgI -mediated enzymatic incorporation into the site opposite templating C is dATP>dTTP≫dCTP, as reported. The net charge, as well as the size and/or shape complementarity of the metal-mediated base pairs, or the stabilities of mismatched base pairs in the absence of metal ions, would be more important than the stability of the metallo-base pairs in the replicating reaction catalyzed by DNA polymerases.

Super-Resolution Imaging of Competitive Unlabeled DNA Hybridization Reveals the Influence of Fluorescent Labels on Duplex Formation and Dissociation Kinetics.

Single-molecule fluorescence imaging is a powerful method to measure reversible reaction kinetics, allowing one to monitor the bound state of individual probe molecules with fluorescently labeled targets. In the case of DNA hybridization, previous studies have shown that the presence of a fluorescent label on a target strand can exhibit significant influence on the stability of a DNA duplex that is formed. In this work, we have developed a super-resolution imaging method to measure the hybridization kinetics of unlabeled target DNA that compete with a fluorescently labeled tracer DNA strand to hybridize with an unlabeled probe DNA immobilized at a surface. The hybridization of an unlabeled DNA target cannot be detected directly, but its presence blocks the immobilized probe DNA, influencing the measured time intervals between labeled DNA hybridization events. We derive a model for competitive hybridization kinetics to extract the association and dissociation rate constants of the unlabeled species from the distribution of time intervals between hybridization events of the labeled tracer DNA at individual localized DNA probe sites. We use this methodology to determine the hybridization kinetics of a model 11-mer unlabeled target DNA strand and then determine how five different fluorescent labels attached to the same target DNA strand impact the hybridization kinetics. Compared to the unlabeled target, these labels can slow the association and dissociation rates by as much as a factor of 5. The super-resolution time-interval methodology provides a unique approach to determining fundamental (label-free) rates of DNA hybridization, revealing the significant influence of fluorescent labels on these kinetics. This measurement concept can be extended to studies of other reversible reaction systems, where kinetics of unlabeled species can be determined from their influence on the reaction of a labeled species with localized probe molecules on a surface.

Structure and Hybridization Properties of Glycine Morpholine Oligomers in Complexes with DNA and RNA: Experimental and Molecular Dynamics Studies.

Methylenecarboxamide (glycine) morpholine oligomers (gMOs) with a modified backbone are new and promising nucleic acid analogues. In this work, a combination of circular dichroism spectroscopy, optical melting, and molecular dynamics simulations was used to investigate hybridization properties of gMOs, as well as the structure and dynamics of their tandem complexes with DNA and RNA. It was shown that the structure of nucleic acids in modified complexes is similar to that of the fully native analogues. The energies of binding and cooperative interactions at the helix-helix interface in the nick were determined experimentally and by computer simulation analysis. Here, we found for the first time, the possibility to determine and predict precisely the thermodynamic parameters of complementary complex formation using the original experimental and computer simulation approaches. It was shown that the use of simulation data in the explicit solvent and the molecular mechanics Poisson-Boltzmann (or generalized Born) surface area methods for the calculation of the hybridization enthalpy makes it possible to evaluate the thermal stability of DNA and gMO tandem duplexes with DNA or RNA with an unexpectedly high accuracy. We found that at high ionic strength and neutral pH, the observed thermal stability of the gMO/RNA tandem complex is similar to that of DNA/DNA and lower than that of gMO/DNA which is close to that of DNA/RNA.

Computational and NMR studies of RNA duplexes with an internal pseudouridine-adenosine base pair

Pseudouridine (Ψ) is the most common chemical modification present in RNA. In general, Ψ increases the thermodynamic stability of RNA. However, the degree of stabilization depends on the sequence and structural context. To explain experimentally observed sequence dependence of the effect of Ψ on the thermodynamic stability of RNA duplexes, we investigated the structure, dynamics and hydration of RNA duplexes with an internal Ψ-A base pair in different nearest-neighbor sequence contexts. The structures of two RNA duplexes containing 5′-GΨC/3′-CAG and 5′-CΨG/3′-GAC motifs were determined using NMR spectroscopy. To gain insight into the effect of Ψ on duplex dynamics and hydration, we performed molecular dynamics (MD) simulations of RNA duplexes with 5′-GΨC/3′-CAG, 5′-CΨG/3′-GAC, 5′-AΨU/3′-UAA and 5′-UΨA/3′-AAU motifs and their unmodified counterparts. Our results showed a subtle impact from Ψ modification on the structure and dynamics of the RNA duplexes studied. The MD simulations confirmed the change in hydration pattern when U is replaced with Ψ. Quantum chemical calculations showed that the replacement of U with Ψ affected the intrinsic stacking energies at the base pair steps depending on the sequence context. The calculated intrinsic stacking energies help to explain the experimentally observed sequence dependent changes in the duplex stability from Ψ modification.

New Oligodeoxynucleotide Derivatives Containing N-(Sulfonyl)-Phosphoramide Groups

New oligodeoxynucleotide derivatives containing N-(o-nitrobenzenesulfonyl)-phosphoramide (nosyl phosphoramide), N-(1-butanesulfonyl)-phosphoramide (busyl phosphoramide), or N-(1-hexanesulfonyl)-phosphoramide groups are described. These compounds were first obtained via solid-phase synthesis on an automatic DNA synthesizer according to the Staudinger reaction between the corresponding sulfonylazides (nosyl, busyl or hesyl azides) and the oligonucleotide containing 3',5'-dinucleoside-β-cyanoethyl phosphide—phosphitamide condensation product—immobilized onto a polymer carrier. In this case, the rate of the Staudinger reaction on the solid phase is higher for more electrophilic nosyl azide than those of less electrophilic busyl and hesyl azides. The nosyl, busyl, and hesyl phosphoramide groups are stable under oligonucleotide synthesis conditions, including acid detritylation and removal of protective groups to form oligonucleotide from a polymer carrier via treatment with concentrated aqueous ammonia at 55°C. The oligonucleotides modified with either nosyl or busyl phosphoramide groups at all internucleotide positions were first prepared. We showed that the stability of complementary duplexes of oligodeoxynucleotides containing busyl or hesyl phosphoramide groups with a single-stranded DNA is insignificantly lower than that of a native DNA:DNA duplex, whereas the destabilization of the duplex is clearer for a bulky hesyl phosphoramide group. The oligonucleotides bearing busyl phosphoramide group form complementary duplexes with a complementary RNA being less stable than native DNA:RNA duplex, but more stable than similar heteroduplexes formed from RNA oligonucleotides and the tosyl phosphoramide group. These DNA N-(sulfonyl)-phosphoramide derivatives are considered to be potential antisense oligonucleotides.

Physicochemical Properties of the Phosphoryl Guanidine Oligodeoxyribonucleotide Analogs

This work describes the basic physicochemical properties of phosphoryl guanidine oligonucleotides (PGOs)—a new type of DNA analog with a partially or totally uncharged backbone. Replacement of the negatively charged oxygen atom with an electroneutral 1,3-dimethylimidazolidine-2-imine (DMI) group is shown to influence the hydrophobicity of oligonucleotide, its electrophoretic mobility, and UV absorption spectra. Thermal stability of DMI-containing DNA-duplexes was studied in aqueous solutions with different ionic strength. It is determined that, in the case of low ionic strength, the presence of DMI groups significantly enhances the thermal stability of the duplex. In the case of nearly physiological condition (0.1 M NaCl, 10 mM MgCl2) or higher ionic strength, DMI groups do not affect the duplex stability or even slightly reduce it (melting temperature changes, on average, by –1.2°C per one DMI group). An important feature of totally uncharged PGO is its ability to form the duplex effectively regardless the ionic strength, even in deionized water. In addition, according to CD data, DMI groups do not disturb the double helix structure of DNA, maintaining it in the B form.

N8-Glycosylated 8-Azapurine and Methylated Purine Nucleobases: Synthesis and Study of Base Pairing Properties.

In this report, we present the synthesis of N8-glycosylated 8-aza-2-methylhypoxanthine and 8-aza-6-thiohypoxanthine 2'-deoxynucleosides as well as methylated 2'-deoxynebularine derivatives. In vitro base pairing properties between each modified and canonical nucleobase were studied. As demonstrated by Tm, incorporation of the modified bases in DNA resulted, with few exceptions, in low stability of duplexes. Modified bases studied in this report are preferentially recognized by T (for N8-glycosylated 8-aza-2-methylhypoxanthine and methylated purines) and G (N8-glycosylated 8-aza-2-methylhypoxanthine). The base pair formed between N8-glycosylated 8-aza-6-thiohypoxanthine and N9-glycosylated 2-methyl-6-thiohypoxanthine (X2:X6) showed, to some extent, an orthogonal interaction. Based on Tm studies, the only potential self-pairing system is formed by the N8-glycosylated 8-aza-6-thiohypoxanthine nucleoside (X2) but only in the absence of canonical G and T. This study indicated that the canonical thymine base is the preferential base partner of methylated purine bases.

Molecular Dynamics Study of the Hybridization between RNA and Modified Oligonucleotides.

MicroRNAs (miRNAs) are attractive drug candidates for many diseases as they can modulate the expression of gene networks. Recently, we discovered that DNAs targeting microRNA-22-3p (miR-22-3p) hold the potential for treating obesity and related metabolic disorders (type 2 diabetes mellitus, hyperlipidemia, and nonalcoholic fatty liver disease (NAFLD)) by turning fat-storing white adipocytes into fat-burning adipocytes. In this work, we explored the effects of chemical modifications, including phosphorothioate (PS), locked nucleic acid (LNA), and peptide nucleic acid (PNA), on the structure and energy of DNA analogs by using molecular dynamics (MD) simulations. To achieve a reliable prediction of the hybridization free energy, the AMOEBA polarizable force field and the free energy perturbation technique were employed. The calculated hybridization free energies are generally compatible with previous experiments. For LNA and PNA, the enhanced duplex stability can be explained by the preorganization mechanism, i.e., the single strands adopt stable helical structures similar to those in the duplex. For PS, the S and R isomers (Sp and Rp) have preferences for C2'-endo and C3'-endo sugar puckering conformations, respectively, and therefore Sp is less stable than Rp in DNA/RNA hybrids. In addition, the solvation penalty of Rp accounts for its destabilization effect. PS-LNA is similar to LNA as the sugar puckering is dominated by the locked sugar ring. This work demonstrated that MD simulations with polarizable force fields are useful for the understanding and design of modified nucleic acids.

Applications of Catalytic Hairpin Assembly Reaction in Biosensing.

Nucleic acids are considered as perfect programmable materials for cascade signal amplification and not merely as genetic information carriers. Among them, catalytic hairpin assembly (CHA), an enzyme-free, high-efficiency, and isothermal amplification method, is a typical example. A typical CHA reaction is initiated by single-stranded analytes, and substrate hairpins are successively opened, resulting in thermodynamically stable duplexes. CHA circuits, which were first proposed in 2008, present dozens of systems today. Through in-depth research on mechanisms, the CHA circuits have been continuously enriched with diverse reaction systems and improved analytical performance. After a short time, the CHA reaction can realize exponential amplification under isothermal conditions. Under certain conditions, the CHA reaction can even achieve 600 000-fold signal amplification. Owing to its promising versatility, CHA is able to be applied for analysis of various markers in vitro and in living cells. Also, CHA is integrated with nanomaterials and other molecular biotechnologies to produce diverse readouts. Herein, the varied CHA mechanisms, hairpin designs, and reaction conditions are introduced in detail. Additionally, biosensors based on CHA are presented. Finally, challenges and the outlook of CHA development are considered.

Effects of metal ions on thermal stabilities of DNA duplexes containing homo- and heterochiral mismatched base pairs: comparison of internal and terminal substitutions

The effects of metal ions on the stabilities of duplexes containing a D-homochiral and heterochiral mismatched base pairs were studied. In some duplexes containing an internal mismatched base pair, significant stabilization by HgII and AgI ions was observed. While, in duplexes containing a terminal mismatched base pair, only the duplexes containing T-T and LT-T mispairs were significantly stabilized by HgII ions, and the stabilities of the duplexes containing T-T and LT-T mispairs exceeded those of the corresponding homochiral matched duplex. The results suggest that the formation of homo- and heterochiral T-HgII-T base pairs at duplex termini would be useful for the thermal and enzymatic stabilization of DNA-based nanodevice.

Duplex-forming Oligonucleotide of Triazole-linked RNA.

An oligonucleotide of triazole-linked RNA (TL RNA) was synthesized by performing consecutive copper-catalyzed azide-alkyne cycloaddition reactions for elongation. The reaction conditions that had been optimized for the synthesis of 3-mer TL RNA were found to be inappropriate for longer oligonucleotides, and the conditions were reoptimized for the solid-phase synthesis of an 11-mer TL RNA oligonucleotide. Duplex formation of the 11-mer TL RNA oligonucleotide was examined with the complementary oligonucleotide of natural RNA to reveal the effects of the 2'-OH groups on the duplex stability.

Thermal Stability of Peptide Nucleic Acid Complexes.

Peptide nucleic acid (PNA) is a neutral nucleic acid analogue that base pairs with itself and natural nucleic acids. PNA-nucleic acid complexes are more thermally stable than the corresponding complexes of natural nucleic acids. In addition, PNA is biostable and thus used in many antisense and antigene applications to block functional RNA or DNA via sequence-specific interactions. We have recently developed force field parameters for molecular dynamics (MD) simulations of PNA and PNA-involving duplexes with natural nucleic acids. In this work, we provide the first application of this force field to biologically relevant PNA sequences and their complexes with RNA. We investigated thermal stabilities of short PNA-PNA, PNA-RNA, and RNA-RNA duplexes using UV-monitored thermal denaturation experiments and MD simulations at ambient and elevated temperatures. The simulations show a two-state melting transition and reproduce the thermal stability from melting experiments, with PNA-PNA being the most and RNA-RNA the least stable. The PNA-PNA duplex also displays the highest activation energy for melting. The atomistic details of unfolding of PNA duplexes suggest that all PNA-PNA bases melt concomitantly, whereas the RNA-RNA and PNA-RNA are destabilized from the termini toward the central part of the duplexes.

Electrochemical voltammetric behaviors of synthetic dengue virus RNAs at ITO sensing electrode

An electroconductive indium tin oxide (ITO) electrode was assembled and used as a sensing platform for direct detection of Dengue virus (DENV) RNAs in the phosphate buffer solution at pH 7.4. The voltammetric behaviors of DENV RNAs were examined at different volume ratio with matched and mismatched RNAs. The stability of the DENV RNAs hybrid duplexes and the microenvironment on the electrode surface was found to affect their voltammetric profiles. In this work, the detection limit was estimated to be 2 amol. No labeling, reverse transcription and/or polymerase chain reaction is required for this RNA detection technology. This simple and efficient ITO sensing electrode may be a promising platform in Dengue diagnosis.

Stabilization of DNA Loop Structures by Large Cations.

The DNA-binding properties of large cations differ from those of metal ions due to steric exclusion from base-paired regions. In this study, the thermal stability of DNA secondary structures, including duplexes, internal loops, bulge loops, hairpin loops, dangling ends, and G-quadruplexes, was investigated in the presence of cations of different sizes. Large cations, such as tetrabutylammonium and tetrapentylammonium ions, reduced the stability of fully matched duplexes but increased the stability of duplexes with a long loop. The cations also increased the stability of G-quadruplexes with a long loop, and the degree of stabilization was greater for low-stability G-quadruplexes. Analysis of the salt concentration dependence indicates that large cations bind to the loop nucleotides, leading to counteracting the destabilization effect on base pairing. It is likely that binding occurs when loop nucleotides are sufficiently flexible to allow for greater accessibility for large cations. These results provide insight into nucleic acid interactions with large cationic molecules and suggest a potential method for stabilizing noncanonical DNA structures under intracellular conditions.

Priming and realignment by the influenza a virus RdRp is dependent on the length of the host primers and the extent of base pairing to viral RNA

Initiation of influenza A virus (IAV) transcription depends on RNA primers derived from host RNAs. During this process, some primers are elongated by a few nucleotides, realigned on the viral RNA templates (vRNA), and then used to initiate another round of transcription. Here, we used information on the host primers used by four IAV strains and four mini-replicons to investigate the characteristics of primer undergoing priming and realignment. We report that primers are biased towards this mechanism on the basis of length and RNA duplex stability with the vRNA templates. Priming and realignment results in primers three nucleotides longer, ending in a nucleotide sequence able to base pair with the 3′ end of the vRNA template. By acting on primers based on length and sequence compatibility with the 3’ end of the vRNA, priming and realignment rescues suboptimal primers, converting them into ones that can efficiently initiate transcription.

Probing Local Folding Allows Robust Metal Sensing Based on a Na+ -Specific DNAzyme.

Fluorescent metal sensors based on DNA often rely on changes in end-to-end distance or local environmental near fluorophore labels. Because metal ions can also nonspecifically interact with DNA through various mechanisms, such as charge screening, base binding, and increase or decrease in duplex stability, robust and specific sensing of metal ions has been quite challenging. In this work, a side-by-side comparison of two signaling strategies on a Na+ -specific DNAzyme that contained a Na+ -binding aptamer was performed. The duplex regions of the DNAzyme was systematically shortened and its effect was studied by using a 2-aminopurine (2AP)-labeled substrate strand. Na+ binding affected the local environmental of the 2AP label and increased its fluorescence. A synergistic process of Na+ binding and forming the duplex on the 5'-end of the enzyme strand was observed, and this end was close to the aptamer loop. Effective Na+ binding was achieved with a five base-pair stem. The effect on the 3'-end is more continuous, and the stem needs to form first before Na+ can bind. With an optimized substrate binding arm, a FRET-based sensor has been designed by labeling the two ends of a cis form of the DNAzyme with two fluorophores. In this case, Na+ failed to show a distinct difference from that of Li+ or K+ ; thus indicating that probing changes to the local environment allows more robust sensing of metal ions.

Effect of Staple Age on DNA Origami Nanostructure Assembly and Stability.

DNA origami nanostructures are widely employed in various areas of fundamental and applied research. Due to the tremendous success of the DNA origami technique in the academic field, considerable efforts currently aim at the translation of this technology from a laboratory setting to real-world applications, such as nanoelectronics, drug delivery, and biosensing. While many of these real-world applications rely on an intact DNA origami shape, they often also subject the DNA origami nanostructures to rather harsh and potentially damaging environmental and processing conditions. Furthermore, in the context of DNA origami mass production, the long-term storage of DNA origami nanostructures or their pre-assembled components also becomes an issue of high relevance, especially regarding the possible negative effects on DNA origami structural integrity. Thus, we investigated the effect of staple age on the self-assembly and stability of DNA origami nanostructures using atomic force microscopy. Different harsh processing conditions were simulated by applying different sample preparation protocols. Our results show that staple solutions may be stored at −20 °C for several years without impeding DNA origami self-assembly. Depending on DNA origami shape and superstructure, however, staple age may have negative effects on DNA origami stability under harsh treatment conditions. Mass spectrometry analysis of the aged staple mixtures revealed no signs of staple fragmentation. We, therefore, attribute the increased DNA origami sensitivity toward environmental conditions to an accumulation of damaged nucleobases, which undergo weaker base-pairing interactions and thus lead to reduced duplex stability.

3-Fluoro-2-mercuri-6-methylaniline Nucleotide as a High-Affinity Nucleobase-Specific Hybridization Probe.

A 3-fluoro-6-methylaniline nucleoside was synthesized and incorporated into an oligonucleotide, and its ability to form mercury-mediated base pairs was studied. UV melting experiments revealed increased duplex stability with thymine, guanine, and cytosine opposite to the probe and a clear nucleobase-specific binding preference (T > G > C > A). Moreover, the 3-fluoro group was utilized as a spin label that showed distinct 19F NMR resonance shifts depending on the complementary nucleobase, providing more detailed information on Hg(II)-mediated base pairing.

High-throughput analysis revealed mutations’ diverging effects on SMN1 exon 7 splicing

ABSTRACT Splicing-affecting mutations can disrupt gene function by altering the transcript assembly. To ascertain splicing dysregulation principles, we modified a minigene assay for the parallel high-throughput evaluation of different mutations by next-generation sequencing. In our model system, all exonic and six intronic positions of the SMN1 gene’s exon 7 were mutated to all possible nucleotide variants, which amounted to 180 unique single-nucleotide mutants and 470 double mutants. The mutations resulted in a wide range of splicing aberrations. Exonic splicing-affecting mutations resulted either in substantial exon skipping, supposedly driven by predicted exonic splicing silencer or cryptic donor splice site (5′ss) and de novo 5′ss strengthening and use. On the other hand, a single disruption of exonic splicing enhancer was not sufficient to cause major exon skipping, suggesting these elements can be substituted during exon recognition. While disrupting the acceptor splice site led only to exon skipping, some 5′ss mutations potentiated the use of three different cryptic 5′ss. Generally, single mutations supporting cryptic 5′ss use displayed better pre-mRNA/U1 snRNA duplex stability and increased splicing regulatory element strength across the original 5′ss. Analyzing double mutants supported the predominating splicing regulatory elements’ effect, but U1 snRNA binding could contribute to the global balance of splicing isoforms. Based on these findings, we suggest that creating a new splicing enhancer across the mutated 5′ss can be one of the main factors driving cryptic 5′ss use.