Thermal Stability Of Duplexes Research Articles

2′-Deoxyribo-oligonucleotides duplexes and G-quadruplexes structures and thermal stability in 1‑butyl‑1-methylpiperidinium and 1-octyl-3-methylimidazolium bromide ionic liquid solutions

Conformation and thermal stability of duplexes made of various ratio of purine and pyrimidine, but also of two G-quadruplexes, were evaluated in two different ionic liquids, aliphatic 1‑butyl‑1-methylpiperidinium bromide and 1-octyl-3-methylimidazolium bromide with an aromatic moiety, by means of UV-spectroscopy and circular dichroism. It appeared that no conformational change occurred within all structures studied whatever the medium was, only thermal stabilities were affected.

Synthesis of 2'-O-Methyl/2'-O-MOE-L-Nucleoside Derivatives and Their Applications: Preparation of G-Quadruplexes, Their Characterization, and Stability Studies.

Nucleosides and their analogues constitute an important family of molecules with potential antiviral and antiproliferative activity. The enantiomers of natural nucleosides, l-nucleoside derivatives, which have comparable biological activity but more favorable toxicological properties and greater metabolic stability than d-nucleosides, have emerged as a new class of therapeutic agents. Furthermore, l-nucleosides can be used as a building block to prepare l-oligonucleotides, which have identical physical properties in terms of solubility, hybridization kinetics, and duplex thermal stability as d-oligonucleotides but completely orthogonal in nature. Consequently, they are resistant to nuclease degradation, nontoxic, and immunologically passive, which are desirable properties for biomedical applications. Herein, we describe the synthesis of several 2'-O-methyl/2'-O-MOE-l-nucleoside pyrimidine derivatives and their incorporation into G-rich oligonucleotides. Finally, we evaluated the stability and resistance against nucleases of these new G-quadruplexes, demonstrating the potential of the l-nucleosides described in this work in providing enhanced nuclease resistance with a minimal impact in the nucleic acid structural properties.

Systematic Investigation of Tether Length and Phosphorus Configuration in Backbone Constrained Macrocyclic Nucleic Acids to Modulate Binding Kinetics for RNA.

We recently described a chemical strategy to pre-organize a trinucleotide subunit in a conformation suitable for Watson-Crick base pairing for modulating the binding kinetics of single-stranded oligonucleotides (ONs) using bis-phosphonate esters bridging hydrocarbon tethers to provide 11- and 15-membered macrocyclic analogues. In this manuscript, we describe the synthesis of all eight P-stereoisomers of macrocyclic 12-, 13-, 14-, and 16-membered hydrocarbon-bridged nucleotide trimers, their incorporation into ONs, and biophysical characterization of the modified ONs. The size of the macrocyclic tether and configuration at phosphorus had profound effects on hybridization kinetics. ONs containing 12- and 13-membered rings exhibited faster on-rates (up to 5-fold) and off-rates (up to 161-fold). In contrast, ONs using the larger ring size macrocycles generally exhibited smaller changes in binding kinetics relative to unmodified DNA. Interestingly, several of the analogues retained significant binding affinity for RNA based on their dissociation constants, despite being modestly destabilizing in the thermal denaturation experiments, highlighting the potential utility of measuring dissociation constants versus duplex thermal stability when evaluating novel nucleic acid analogues. Overall, our results provide additional insights into the ability of backbone-constrained macrocyclic nucleic acid analogues to modulate hybridization kinetics of modified ONs with RNA.

Experimental and theoretical rationalization for the base pairing abilities of inosine, guanosine, adenosine, and their corresponding 8-oxo-7,8-dihydropurine, and 8-bromopurine analogues within A-form duplexes of RNA.

Inosine is an important RNA modification, furthermore RNA oxidation has gained interest due, in part, to its potential role in the development/progression of disease as well as on its impact on RNA structure and function. In this report we established the base pairing abilities of purine nucleobases G, I, A, as well as their corresponding, 8‐oxo‐7,8‐dihydropurine (common products of oxidation at the C8‐position of purines), and 8‐bromopurine (as probes to explore conformational changes), derivatives, namely 8‐oxoG, 8‐oxoI, 8‐oxoA, 8‐BrG, and 8‐BrI. Dodecamers of RNA were obtained using standard phosphoramidite chemistry via solid‐phase synthesis, and used as models to establish the impact that each of these nucleobases have on the thermal stability of duplexes, when base pairing to canonical and noncanonical nucleobases. Thermal stabilities were obtained from thermal denaturation transition (T m) measurements, via circular dichroism (CD). The results were then rationalized using models of base pairs between two monomers, via density functional theory (DFT), that allowed us to better understand potential contributions from H‐bonding patterns arising from distinct conformations. Overall, some of the important results indicate that: (a) an anti‐I:syn‐A base pair provides thermal stability, due to the absence of the exocyclic amine; (b) 8‐oxoG base pairs like U, and does not induce destabilization within the duplex when compared to the pyrimidine ring; (c) a U:G wobble‐pair is only stabilized by G; and (d) 8‐oxoA displays an inherited base pairing promiscuity in this sequence context. Gaining a better understanding of how this oxidatively generated lesions potentially base pair with other nucleobases will be useful to predict various biological outcomes, as well as in the design of biomaterials and/or nucleotide derivatives with biological potential.

Synthesis, chirality-dependent conformational and biological properties of siRNAs containing 5'-(R)- and 5'-(S)-C-methyl-guanosine.

Various chemical modifications have been identified that enhance potency of small interfering RNAs (siRNAs) and that reduce off-target effects, immune stimulation, and toxicities of metabolites of these therapeutic agents. We previously described 5′-C-methyl pyrimidine nucleotides also modified at the 2′ position of the sugar. Here, we describe the synthesis of 2′-position unmodified 5′-(R)- and 5′-(S)-C-methyl guanosine and evaluation of these nucleotides in the context of siRNA. The (R) isomer provided protection from 5′ exonuclease and the (S) isomer provided protection from 3′ exonuclease in the context of a terminally modified oligonucleotide. siRNA potency was maintained when these modifications were incorporated at the tested positions of sense and antisense strands. Moreover, the corresponding 5′ triphosphates were not substrates for mitochondrial DNA polymerase. Models generated based on crystal structures of 5′ and 3′ exonuclease oligonucleotide complexes with 5′-(R)- and 5′-(S)-C-methyl substituents attached to the 5′- and 3′-terminal nucleotides, respectively, provided insight into the origins of the observed protections. Structural properties of 5′-(R)-C-methyl guanosine incorporated into an RNA octamer were analysed by X-ray crystallography, and the structure explains the loss in duplex thermal stability for the (R) isomer compared with the (S) isomer. Finally, the effect of 5′-C-methylation on endoribonuclease activity has been explained.

The Effect of Divalent Metal Ions on the Temperature Stability of Poly(I:C) Duplex

The effect of divalent ions on the thermal stability of the double-stranded polynucleotide polyinosinic:polycytidylic acid (poly(I:C)) in a buffer solution (pH 7) was studied by UV absorption spectroscopy and thermal denaturation by measuring the absorbance at 260 nm. The concentration dependence of the polynucleotide melting temperature (Tm) in the presence of metal ions (2.5 × 10−6–10−2 M) was obtained. It was found that the duplex thermal stability grew gradually upon an increase of Ca2+ and Mn2+ concentration. The thermal stability of the duplex in the presence of Cd2+, Ni2+, and Zn2+ rises up to an ion concentration of about 10−4 M and further falls. In contrast to these ions, the addition of Cu2+ to the solution leads to a sharp decline in Тm of the duplex at a concentration above 10−5 M. The observed difference in the influence of metal ions on the poly(I:C) thermal stability is explained by the different affinity of these ions to both the phosphate groups and the nitrogenous bases of the polynucleotides. Based on the analysis of the Tm concentration dependence, the dominant binding of each ion to the specific polynucleotide components was proposed.

Mutually stabilizing interactions between proto-peptides and RNA

The close synergy between peptides and nucleic acids in current biology is suggestive of a functional co-evolution between the two polymers. Here we show that cationic proto-peptides (depsipeptides and polyesters), either produced as mixtures from plausibly prebiotic dry-down reactions or synthetically prepared in pure form, can engage in direct interactions with RNA resulting in mutual stabilization. Cationic proto-peptides significantly increase the thermal stability of folded RNA structures. In turn, RNA increases the lifetime of a depsipeptide by >30-fold. Proto-peptides containing the proteinaceous amino acids Lys, Arg, or His adjacent to backbone ester bonds generally promote RNA duplex thermal stability to a greater magnitude than do analogous sequences containing non-proteinaceous residues. Our findings support a model in which tightly-intertwined biological dependencies of RNA and protein reflect a long co-evolutionary history that began with rudimentary, mutually-stabilizing interactions at early stages of polypeptide and nucleic acid co-existence.

Double-headed nucleotides as xeno nucleic acids: information storage and polymerase recognition.

Xeno nucleic acids (XNAs) are artificial genetic systems based on sugar-modified nucleotides. Herein, we investigate double-headed nucleotides as a new XNA. A new monomer, AT, is presented, and together with previous double-headed nucleotide monomers, new nucleic acid motifs consisting of up to five consecutive A·T base pairs have been obtained. Sections composed entirely of double-headed nucleotides are well-tolerated within a DNA duplex and can condense the genetic information. For instance, a 13-mer duplex is condensed to an 11-mer modified duplex containing four double-headed nucleotides while simultaneously improving duplex thermal stability with +14.0 °C. Also, the transfer of information from double-headed to natural nucleotides by DNA polymerases has been examined. The first double-headed nucleoside triphosphate was prepared but could not be recognized and incorporated by the tested DNA polymerases. On the other hand, it proved possible for Therminator DNA polymerase to transfer the information of a double-headed nucleotide in a template sequence to natural DNA under controlled conditions.

Aflatoxin B₁⁻Formamidopyrimidine DNA Adducts: Relationships between Structures, Free Energies, and Melting Temperatures.

Thermal stabilities of DNA duplexes containing Gua (g), α- (a) or β-anomer of formamidopyrimidine-N7-9-hydroxy-aflatoxin B1 (b) differ markedly (Tm: ), but the underlying molecular origin of this experimentally observed phenomenon is yet to be identified and determined. Here, by employing explicit-solvent molecular dynamics simulations coupled with free-energy calculations using a combined linear-interaction-energy/linear-response-approximation approach, we explain the quantitative differences in T in terms of three structural features (bulkiness, order, and compactness) and three energetical contributions (non-polar, electrostatic, and preorganized-electrostatic), and thus advance the current understanding of the relationships between structures, free energies, and thermal stabilities of DNA double helices.

Oligonucleotide Primers with G8AE-Clamp Modifications for RT-qPCR Detection of the Low-Copy dsRNA.

We developed a new technique suitable for improved detection of low-copy dsRNA using modified oligonucleotides as primers in RT-qPCR. Insertion of G8AE-clamp residues into primers significantly improves thermal stability of duplexes with RNA without decrease of hybridization selectivity. The applicability of modified primers is demonstrated for detection of low-copy Kemerovo virus dsRNA.

Structural Studies and Gene Silencing Activity of siRNAs Containing Cationic Phosphoramidate Linkages.

A series of siRNA duplexes containing cationic non-bridging 3',5'-linked phosphoramidate (PN) linkages was designed and synthesized using a combination of phosphoramidite and H-phosphonate chemistries. Modified oligonucleotides were assayed for their thermal stability, helical structure, and ability to modulate the expression of firefly luciferase. We demonstrate that PN modifications of siRNAs are, in general, minimally destabilizing with respect to duplex thermal stability; destabilization can be mitigated through the incorporation of 2'-modified RNA-like residues or PN conjugates containing ionizable pendant moieties. We also demonstrate that single cationic dimethylethylenediamine PN linkages have little effect on siRNA potency, whether located in the passenger or guide strand of the duplex. Highly modified siRNA passenger strands were further modified with up to four cationic PN linkages, with little effect on duplex potency or helical structure. We envision that PN modifications could be useful in the production of therapeutic siRNAs with optimal biological properties.

Deformability Calculation for Estimation of the Relative Stability of Chemically Modified RNA Duplexes.

Chemical modification of RNA duplexes alters their stability. We have attempted to develop a computational approach to estimate the thermal stability of chemically modified duplexes. These studies revealed that the deformability of chemically modified RNA duplexes, calculated from molecular dynamics simulations, could be used as a good indicator for estimating the effect of chemical modification on duplex thermal stability. This unit describes how deformability calculation can be applied to estimate the relative stability of chemically modified RNA duplexes. © 2017 by John Wiley & Sons, Inc.

Synthesis and Biophysical Investigations of Oligonucleotides Containing Galactose-Modified DNA, LNA, and 2'-Amino-LNA Monomers.

Galactose-modified thymidine, LNA-T, and 2'-amino-LNA-T nucleosides were synthesized, converted into the corresponding phosphoramidite derivatives and introduced into short oligonucleotides. Compared to the unmodified control strands, the galactose-modified oligonucleotides in general, and the N2'-functionalized 2'-amino-LNA derivatives in particular, showed improved duplex thermal stability against DNA and RNA complements and increased ability to discriminate mismatches. In addition, the 2'-amino-LNA-T derivatives induced remarkable 3'-exonuclease resistance. These results were further investigated using molecular modeling studies.

Lipophilic 2'-O-Acetal Ester RNAs: Synthesis, Thermal Duplex Stability, Nuclease Resistance, Cellular Uptake, and siRNA Activity after Spontaneous Naked Delivery.

The in vivo application of siRNA depends on its cellular uptake and intracellular release, and this is an unsatisfactorily resolved technical hurdle in medicinal applications. Promising concepts directed towards providing efficient cellular and intracellular delivery include lipophilic chemical modification of siRNA. Here we describe chemistry for the production of modified siRNAs designed to display improved transmembrane transport into human cells while preserving the potency of the RNAi-based inhibitors. We report the synthesis and the biochemical and biophysical characteristics of 2'-O-phenylisobutyryloxymethyl (PiBuOM)-modified siRNAs and their impact on biological activity. In the case of spontaneous cellular uptake of naked PiBuOM-modified siRNA, we observed increased target suppression in human cells relative to unmodified or pivaloyloxymethyl (PivOM)-modified siRNA. We provide evidence of improved spontaneous cellular uptake of naked PiBuOM-modified siRNA and of substantial target suppression in human cells in serum-containing medium.

Hybridization accompanying FRET event in labeled natural nucleoside-unnatural nucleoside containing chimeric DNA duplexes

Förster resonance energy transfer (FRET) is a highly efficient strategy in illuminating the structures, structural changes and dynamics of DNA, proteins and other biomolecules and thus is being widely utilized in studying such phenomena, in designing molecular/biomolecular probes for monitoring the hybridization event of two single stranded DNA to form duplex, in gene detection and in many other sensory applications in chemistry, biology and material sciences. Moreover, FRET can give information about the positional status of chromophores within the associated biomolecules with much more accuracy than other methods can yield. Toward this end, we want to report here the ability of fluorescent unnatural nucleoside, triazolylphenanthrene (TPhenBDo) to show FRET interaction upon hybridization with fluorescently labeled natural nucleosides, PerU or OxoPyU or PerU, forming two stable chimeric DNA duplexes. The pairing selectivity and the thermal duplex stability of the chimeric duplexes are higher than any of the duplexes with natural nucleoside formed. The hybridization results in a Förster resonance energy transfer (FRET) from donor triazolylphenanthrene of TPhenBDo to acceptor oxopyrene of OxoPyU and/or to perylene chromophore of PerU, respectively, in two chimeric DNA duplexes. Therefore, we have established the FRET process in two chimeric DNA duplexes wherein a fluorescently labeled natural nucleoside (OxoPyU or PerU) paired against an unnatural nucleoside (TPhenBDo) without sacrificing the duplex stability and B-DNA conformation. The hybridization accompanying FRET event in these classes of interacting fluorophores is new. Moreover, there is no report of such designed system of chimeric DNA duplex. Our observed phenomenon and the design can potentially be exploited in designing more of such efficient FRET pairs for useful application in the detection and analysis of biomolecular interactions and in material science application.

The formation of catalytically competent enzyme–substrate complex is not a bottleneck in lesion excision by human alkyladenine DNA glycosylase

Human alkyladenine DNA glycosylase (AAG) protects DNA from alkylated and deaminated purine lesions. AAG flips out the damaged nucleotide from the double helix of DNA and catalyzes the hydrolysis of the N-glycosidic bond to release the damaged base. To understand better, how the step of nucleotide eversion influences the overall catalytic process, we performed a pre-steady-state kinetic analysis of AAG interaction with specific DNA-substrates, 13-base pair duplexes containing in the 7th position 1-N6-ethenoadenine (εA), hypoxanthine (Hx), and the stable product analogue tetrahydrofuran (F). The combination of the fluorescence of tryptophan, 2-aminopurine, and 1-N6-ethenoadenine was used to record conformational changes of the enzyme and DNA during the processes of DNA lesion recognition, damaged base eversion, excision of the N-glycosidic bond, and product release. The thermal stability of the duplexes characterized by the temperature of melting, Tm, and the rates of spontaneous opening of individual nucleotide base pairs were determined by NMR spectroscopy. The data show that the relative thermal stability of duplexes containing a particular base pair in position 7, (Tm(F/T) < Tm(εA/T) < Tm(Hx/T) < Tm(A/T)) correlates with the rate of reversible spontaneous opening of the base pair. However, in contrast to that, the catalytic lesion excision rate is two orders of magnitude higher for Hx-containing substrates than for substrates containing εA, proving that catalytic activity is not correlated with the stability of the damaged base pair. Our study reveals that the formation of the catalytically competent enzyme–substrate complex is not the bottleneck controlling the catalytic activity of AAG.

Base pairing and structural insights into the 5-formylcytosine in RNA duplex.

5-Formylcytidine (f5C), a previously discovered natural nucleotide in the mitochondrial tRNA of many species including human, has been recently detected as the oxidative product of 5-methylcytidine (m5C) through 5-hydroxymethylcytidine (hm5C) in total RNA of mammalian cells. The discovery indicated that these cytosine derivatives in RNA might also play important epigenetic roles similar as in DNA, which has been intensively investigated in the past few years. In this paper, we studied the base pairing specificity of f5C in different RNA duplex contexts. We found that the 5-formyl group could increase duplex thermal stability and enhance base pairing specificity. We present three high-resolution crystal structures of an octamer RNA duplex [5′-GUA(f5C)GUAC-3′]2 that have been solved under three crystallization conditions with different buffers and pH values. Our results showed that the 5-formyl group is located in the same plane as the cytosine base and forms an intra-residue hydrogen bond with the amino group in the N4 position. In addition, this modification increases the base stacking between the f5C and the neighboring bases while not causing significant global and local structure perturbations. This work provides insights into the effects of 5-formylcytosine on RNA duplex.

Effect of backbone chemistry on hybridization thermodynamics of oligonucleic acids: a coarse-grained molecular dynamics simulation study.

In this paper we study how varying oligonucleic acid backbone chemistry affects the hybridization/melting thermodynamics of oligonucleic acids. We first describe the coarse-grained (CG) model with tunable parameters that we developed to enable the study of both naturally occurring oligonucleic acids, such as DNA, and their chemically-modified analogues, such as peptide nucleic acids (PNAs) and locked nucleic acids (LNAs). The DNA melting curves obtained using such a CG model and molecular dynamics simulations in an implicit solvent and with explicit ions match with the melting curves obtained using the empirical nearest-neighbor models. We use these CG simulations to then elucidate the effect of backbone flexibility, charge, and nucleobase spacing along the backbone on the melting curves, potential energy and conformational entropy change upon hybridization and base-pair hydrogen bond residence time. We find that increasing backbone flexibility decreases duplex thermal stability and melting temperature mainly due to increased conformational entropy loss upon hybridization. Removing charges from the backbone enhances duplex thermal stability due to the elimination of electrostatic repulsion and as a result a larger energetic gain upon hybridization. Lastly, increasing nucleobase spacing decreases duplex thermal stability due to decreasing stacking interactions that are important for duplex stability.

Three new double-headed nucleotides with additional nucleobases connected to C-5 of pyrimidines; synthesis, duplex and triplex studies

In the search for double-coding DNA-systems, three new pyrimidine nucleosides, each coded with an additional nucleobase anchored to the major groove face, are synthesized. Two of these building blocks carry a thymine at the 5-position of 2′-deoxyuridine through a methylene linker and a triazolomethylene linker, respectively. The third building block carries an adenine at the 6-position of pyrrolo-2′-deoxycytidine through a methylene linker. These double-headed nucleosides are introduced into oligonucleotides and their effects on the thermal stabilities of duplexes are studied. All studied double-headed nucleotide monomers reduce the thermal stability of the modified duplexes, which is partially compensated by using consecutive incorporations of the modified monomers or by flanking the new double-headed analogs with members of our former series containing propyne linkers. Also their potential in triplex-forming oligonucleotides is studied for two of the new double-headed nucleotides as well as the series of analogs with propyne linkers. The most stable triplexes are obtained with single incorporations of additional pyrimidine nucleobases connected via the propyne linker.

Thermal stability and conformation of antiparallel duplexes formed by P-stereodefined phosphorothioate DNA/LNA chimeric oligomers with DNA and RNA matrices.

3'-O-(2-Thio-4,4-pentamethylene-1,3,2-oxathiaphospholane) derivatives of LNA-type nucleosides (LNA-OTPs, 2a-d; B' = Thy, Ade(Bz), Cyt(Bz), Gua(dmf), respectively) were synthesized and separated into pure P-diastereomers. X-ray analysis allowed for assignment of the absolute configuration of the phosphorus atom in the detritylated, fast-eluting diastereomer 2a. The diastereomerically pure LNA-OTP monomers were used in solid phase synthesis of P-stereodefined chimeric PS-(DNA/LNA) 11-mers containing 2-3 LNA units. Formally, among the phosphorothioate oligomers the biggest enhancement in thermal stability of Watson-Crick paired duplexes was found for [SP-PS]-(DNA/LNA)/RNA duplexes (on average 8.2 °C per LNA nucleotide), followed by [RP-PS]-(DNA/LNA)/RNA (6.3 °C), [RP-PS]-(DNA/LNA)/DNA (3.8 °C) and [SP-PS]-(DNA/LNA)/DNA (2.4 °C per LNA nucleotide). However, detailed analysis of the thermal dissociation data showed that the thermal stability of (PS-LNA)-containing duplexes does not depend on the spatial orientation of the sulfur atoms. This conclusion received support from CD measurements.