Probing the Binding Interactions between Chemically Modified siRNAs and Human Argonaute 2 Using Microsecond Molecular Dynamics Simulations

The use of chemical modifications in small interfering RNAs (siRNAs) is warranted to impart drug-like properties. However, certain chemical modifications especially those on the sugar have deleterious effects on the RNA interference (RNAi) when they are placed at key positions in the seed region of an siRNA guide strand. In order to probe the effect of chemically modified siRNAs [(2′-<i>O</i>-methyl, 4′-<i>C</i>-aminomethyl-2′-<i>O</i>-methyl, 2′-<i>O</i>-(2-methoxyethyl), and 2′-<i>O</i>-benzyl] on human Argonaute 2 (hAGO2), the catalytic engine of RNAi, we have developed a model of its open conformation. Results from microsecond MD simulations of 15 different siRNA−hAGO2 complexes provide insights about how the key noncovalent interactions and conformational changes at the seed region are modulated, depending upon the nature and position of chemical modifications. Such modification induced structural changes can affect siRNA loading into hAGO2, which may influence RNAi activity. Our studies show that microsecond MD simulations can provide useful information for the design of therapeutically relevant siRNAs

Influence of 2′-Fluoro versus 2′‑<i>O</i>‑Methyl Substituent on the Sugar Puckering of 4′‑<i>C</i>‑Aminomethyluridine

Herein, we report the synthesis of 4′-<i>C</i>-aminomethyl-2′-deoxy-2′-fluorouridine, a therapeutically appealing RNA modification. Conformational analysis by DFT calculations and molecular dynamics simulations using trinucleotide model systems revealed that modified sugar adopts C3′-endo conformation. In this conformer, a weak intramolecular C–H···F H-bond between the hydrogen atom of the 4′-<i>C</i>-CH₂ group and the F atom at the 2′ position is observed. Comparative studies with unmodified, 2′-fluoro-, 2′-<i>O</i>-methyl-, and 4′-<i>C</i>-aminomethyl-2′-<i>O</i>-methyluridine showed the chemical nature of 2′-substituent dictates the sugar puckering of 2′,4′-modified nucleotides

Synthesis and Polymerase-Mediated Bypass Studies of the <i>N</i>²‑Deoxyguanosine DNA Damage Caused by a Lucidin Analogue

Lucidin is a genotoxic and mutagenic hydroxyanthraquinone metabolite, which originates from the roots of Rubia tinctorum L. (madder root). It reacts with exocyclic amino groups of DNA nucleobases and forms adducts/lesions leading to carcinogenesis. To study the effect of lucidin-induced DNA damage, herein, we report the first synthesis of a structural analogue of lucidin [<i>N</i>²-methyl-(1,3-dimethoxyanthraquinone)-deoxyguanosine, LdG] embedded DNAs utilizing phosphoramidite strategy. LdG modification in a DNA duplex imparts destabilization (Δ<i>T</i>_m ∼5 °C/modification), which is attributed to the unfavorable contribution from the enthalpy. Primer extension studies using the Klenow fragment (exo^–) of Escherichia coli DNA polymerase I demonstrate that bypass of LdG modification is error prone as well as slow compared to that across the unmodified sites. Molecular dynamics simulations of the binary complex of Bacillus fragment polymerase (homologue of the Klenow fragment) and LdG-DNA duplex elucidate the structural fluctuations imparted by the LdG lesion, as well as the molecular mechanism of bypass at the lesion site. Overall, the results presented here show that the lucidin adduct destabilizes DNA structure and reduces fidelity and processivity of DNA synthesis

Selective G-quadruplex DNA Stabilizing Agents Based on Bisquinolinium and Bispyridinium Derivatives of 1,8-Naphthyridine

Various biologically relevant G-quadruplex DNA structures offer a platform for therapeutic intervention for altering the gene expression or by halting the function of proteins associated with telomeres. One of the prominent strategies to explore the therapeutic potential of quadruplex DNA structures is by stabilizing them with small molecule ligands. Here we report the synthesis of bisquinolinium and bispyridinium derivatives of 1,8-naphthyridine and their interaction with human telomeric DNA and promoter G-quadruplex forming DNAs. The interactions of ligands with quadruplex forming DNAs were studied by various biophysical, biochemical, and computational methods. Results indicated that bisquinolinium ligands bind tightly and selectively to quadruplex DNAs at low ligand concentration (∼0.2–0.4 μM). Furthermore, thermal melting studies revealed that ligands imparted higher stabilization for quadruplex DNA (an increase in the <i>T</i>_m of up to 21 °C for human telomeric G-quadruplex DNA and >25 °C for promoter G-quadruplex DNAs) than duplex DNA (Δ<i>T</i>_m ≤ 1.6 °C). Molecular dynamics simulations revealed that the end-stacking binding mode was favored for ligands with low binding free energy. Taken together, the results indicate that the naphthyridine-based ligands with quinolinium and pyridinium side chains form a promising class of quadruplex DNA stabilizing agents having high selectivity for quadruplex DNA structures over duplex DNA structures

Thioflavin T as an Efficient Inducer and Selective Fluorescent Sensor for the Human Telomeric G‑Quadruplex DNA

The quest for a G-quadruplex specific fluorescent sensor among other DNA forms under physiological salt conditions has been addressed in this article. We demonstrate for the first time the application of a water-soluble fluorogenic dye, Thioflavin T (ThT), in a dual role of exclusively inducing quadruplex folding in the 22AG human telomeric DNA, both in the presence and absence of Tris buffer/salt, and sensing the same through its fluorescence light-up having emission enhancement of the order of 2100-fold in the visible region. Appropriate conditions allow an apparent switch over of the parallel quadruplex structure in 22AG–ThT (50 mM Tris, pH 7.2) solution to the antiparallel form just by the addition of K⁺ ions in the range 10–50 mM. Moreover, addition of ThT cooperatively stabilizes the K⁺ induced antiparallel quadruplexes by a Δ<i>T</i>_m ∼11 °C. The distinction of ThT as a quadruplex inducer has been contrasted with the erstwhile used structurally related dye, Thiazole Orange (TO), which did not induce any quadruplex folding in the 22AG strand in the absence of salt. The striking fluorescence light-up in ThT on binding to the human telomeric G-quadruplex is shown to be highly specific compared to the less than 250-fold enhancement observed with other single/double strand DNA forms. This work has implication in designing new generation dyes based on the ThT scaffold, which are highly selective for telomeric DNA, for potential diagnostic, therapeutic, and ion-sensing applications

Specific Stabilization of <i>c‑MYC</i> and <i>c‑KIT</i> G-Quadruplex DNA Structures by Indolylmethyleneindanone Scaffolds

Stabilization of G-quadruplex DNA structures by small molecules has emerged as a promising strategy for the development of anticancer drugs. Since G-quadruplex structures can adopt various topologies, attaining specific stabilization of a G-quadruplex topology to halt a particular biological process is daunting. To achieve this, we have designed and synthesized simple structural scaffolds based on an indolylmethyleneindanone pharmacophore, which can specifically stabilize the parallel topology of promoter quadruplex DNAs (<i>c-MYC</i>, <i>c-KIT1</i>, and <i>c-KIT2</i>), when compared to various topologies of telomeric and duplex DNAs. The lead ligands (<b>InEt2</b> and <b>InPr2</b>) are water-soluble and meet a number of desirable criteria for a small molecule drug. Highly specific induction and stabilization of the <i>c-MYC</i> and <i>c-KIT</i> quadruplex DNAs (Δ<i>T</i>_1/2 up to 24 °C) over telomeric and duplex DNAs (Δ<i>T</i>_1/2 ∼ 3.2 °C) by these ligands were further validated by isothermal titration calorimetry and electrospray ionization mass spectrometry experiments (<i>K</i>_a ∼ 10⁵ to 10⁶ M^–1). Low IC₅₀ (∼2 μM) values were emerged for these ligands from a <i>Taq</i> DNA polymerase stop assay with the <i>c-MYC</i> quadruplex forming template, whereas the telomeric DNA template showed IC₅₀ values >120 μM. Molecular modeling and dynamics studies demonstrated the 5′- and 3′-end stacking modes for these ligands. Overall, these results demonstrate that among the >1000 quadruplex stabilizing ligands reported so far, the indolylmethyleneindanone scaffolds stand out in terms of target specificity and structural simplicity and therefore offer a new paradigm in topology specific G-quadruplex targeting for potential therapeutic and diagnostic applications

The <i>N</i>²‑Furfuryl-deoxyguanosine Adduct Does Not Alter the Structure of B‑DNA

<i>N</i>²-Furfuryl-deoxyguanosine (fdG) is carcinogenic DNA adduct that originates from furfuryl alcohol. It is also a stable structural mimic of the damage induced by the nitrofurazone family of antibiotics. For the structural and functional studies of this model <i>N</i>²-dG adduct, reliable and rapid access to fdG-modified DNAs are warranted. Toward this end, here we report the synthesis of fdG-modified DNAs using phosphoramidite chemistry involving only three steps. The functional integrity of the modified DNA has been verified by primer extension studies with DNA polymerases I and IV from <i>E. coli</i>. Introduction of fdG into a DNA duplex decreases the <i>T</i>_m by ∼1.6 °C/modification. Molecular dynamics simulations of a DNA duplex bearing the fdG adduct revealed that though the overall B-DNA structure is maintained, this lesion can disrupt W–C H-bonding, stacking interactions, and minor groove hydrations to some extent at the modified site, and these effects lead to slight variations in the local base pair parameters. Overall, our studies show that fdG is tolerated at the minor groove of the DNA to a better extent compared with other bulky DNA damages, and this property will make it difficult for the DNA repair pathways to detect this adduct

Synthesis, Gene Silencing, and Molecular Modeling Studies of 4′-<i>C</i>-Aminomethyl-2′-<i>O</i>-methyl Modified Small Interfering RNAs

The linear syntheses of 4′-<i>C</i>-aminomethyl-2′-<i>O</i>-methyl uridine and cytidine nucleoside phosphoramidites were achieved using glucose as the starting material. The modified RNA building blocks were incorporated into small interfering RNAs (siRNAs) by employing solid phase RNA synthesis. Thermal melting studies showed that the modified siRNA duplexes exhibited slightly lower <i>T</i>_m (∼1 °C/modification) compared to the unmodified duplex. Molecular dynamics simulations revealed that the 4′-<i>C</i>-aminomethyl-2′-<i>O</i>-methyl modified nucleotides adopt <i>South</i>-type conformation in a siRNA duplex, thereby altering the stacking and hydrogen-bonding interactions. These modified siRNAs were also evaluated for their gene silencing efficiency in HeLa cells using a luciferase-based reporter assay. The results indicate that the modifications are well tolerated in various positions of the passenger strand and at the 3′ end of the guide strand but are less tolerated in the seed region of the guide strand. The modified siRNAs exhibited prolonged stability in human serum compared to unmodified siRNA. This work has implications for the use of 4′-<i>C</i>-aminomethyl-2′-<i>O</i>-methyl modified nucleotides to overcome some of the challenges associated with the therapeutic utilities of siRNAs

4′‑<i>C</i>‑Acetamidomethyl-2′‑<i>O</i>‑methoxyethyl Nucleic Acid Modifications Improve Thermal Stability, Nuclease Resistance, Potency, and hAgo2 Binding of Small Interfering RNAs

In this study, we designed the 4′-C-acetamidomethyl-2′-O-methoxyethyl (4′-C-ACM-2′-O-MOE) uridine and thymidine modifications, aiming to test them into small interfering RNAs. Thermal melting studies revealed that incorporating a single 4′-C-ACM-2′-O-MOE modification in the DNA duplex reduced thermal stability. In contrast, an increase in thermal stability was observed when the modification was introduced in DNA:RNA hybrid and in siRNAs. Thermal destabilization in DNA duplex was attributed to unfavorable entropy, which was mainly compensated by the enthalpy factor to some extent. A single 4′-C-ACM-2′-O-MOE thymidine modification at the penultimate position of the 3′-end of dT20 oligonucleotides in the presence of 3′-specific exonucleases, snake venom phosphodiesterase (SVPD), demonstrated significant stability as compared to monomer modifications including 2′-O-Me, 2′-O-MOE, and 2′-F. In gene silencing studies, we found that the 4′-C-ACM-2′-O-MOE uridine or thymidine modifications at the 3′-overhang in the passenger strand in combination with two 2′-F modifications exhibited superior RNAi activity. The results suggest that the dual modification is well tolerated at the 3′-end of the passenger strand, which reflects better siRNA stability and silencing activity. Interestingly, 4′-C-ACM-2′-O-MOE-modified siRNAs showed considerable gene silencing even after 96 h posttransfection; it showed that our modification could induce prolonged gene silencing due to improved metabolic stability. Molecular modeling studies revealed that the introduction of the 4′-C-ACM-2′-O-MOE modification at the 3′-end of the siRNA guide strand helps to anchor the strand within the PAZ domain of the hAgo2 protein. The overall results indicate that the 4′-C-ACM-2′-O-MOE uridine and thymidine modifications are promising modifications to improve the stability, potency, and hAgo2 binding of siRNAs