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

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