4'-C-Acetamidomethyl-2'-O-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.

N3-Methyluridine and 2'-O-Alkyl/2'-Fluoro-N3-methyluridine functionalized nucleic acids improve nuclease resistance while maintaining duplex geometry.

Herein, we report the synthesis of 2'-O-alkyl/2'-fluoro-N3-methyluridine (2'-O-alkyl/2'-F-m3U) phosphoramidites and their incorporation in DNA and RNA oligonucleotides. The duplex binding affinity and base discrimination studies showed that all 2'-O-alkyl/2'-F-m3U modifications significantly decreased the thermal stability and base-pairing discrimination ability. Serum stability study of dT20 with 2'-O-alkyl-m3U modification exhibited excellent nuclease resistance when incubated with 3'-exonucleases (SVPD) or 5'-exonucleases (PDE-II) as compared to m3U, 2'-F, 2'-OMe modified oligonucleotides. MD simulation studies with RNA tetradecamer duplexes illustrated that the m3U and 2'-O-methyl-m3U modifications reduce the duplex stabilities by disrupting the Watson-Crick hydrogen bonding and base-stacking interactions. Further molecular modelling investigations demonstrated that the 2'-O-propyl-m3U modification exhibits steric interactions with amino acid residues in the active site of 3'- and 5'-exonuclease, leading to enhanced stability. These combined data indicate that the 2'-modified-m3U nucleotides can be used as a promising tool to enhance the stability, silencing efficiency, and drug-like properties of antisense/siRNA-based therapeutics.

Vinyl substituted triphenylamine based turn-off fluorescent probe for selective and sensitive detection of mercury (II) in water and live cells.

Here, we report vinyl substituted triphenylamine (TPA-alk) fluorescent probe for the rapid and efficient detection of mercury ion (Hg2+) in water and biological environment. TPA-alk detects Hg2+ selectively over a wide range of competitive metal ions with a blue shift of 43 nm in the UV absorbance spectrum. The detection limit is found to be 0.146 µM (29.2 ppb) with high selectivity over a wide range of competitive metal ions. DFT study explains the blue shift in the UV-vis absorption band of the optical probe upon the addition of Hg2+. Cell viability assay illustrates that the probe is biocompatible and it has low cytotoxicity even at its higher concentration. Cell imaging studies demonstrate the efficiency of the TPA-alk probe for the micromolar detection of mercury (II) in live BMG1 cells.

Influence of Sugar Modifications on the Nucleoside Conformation and Oligonucleotide Stability: A Critical Review.

Ribofuranose sugar conformation plays an important role in the structure and dynamics of functional nucleic acids such as siRNAs, AONs, aptamers, miRNAs, etc. To improve their therapeutic potential, several chemical modifications have been introduced into the sugar moiety over the years. The stability of the oligonucleotide duplexes as well as the formation of stable and functional protein-oligonucleotide complexes are dictated by the conformation and dynamics of the sugar moiety. In this review, we systematically categorise various ribofuranose sugar modifications employed in DNAs and RNAs so far. We discuss different stereoelectronic effects imparted by different substituents on the sugar ring and how these effects control sugar puckering. Using this data, it would be possible to predict the precise use of chemical modifications and design novel sugar-modified nucleosides for therapeutic oligonucleotides that can improve their physicochemical properties.

Advances in siRNA therapeutics and synergistic effect on siRNA activity using emerging dual ribose modifications.

Nucleic acid-based therapeutics that control gene expression have been steadily progressing towards achieving their full clinical potential throughout the last few decades. Rapid progress has been achieved in RNAi-based therapy by optimizing high specificity and gene silencing efficiency using chemically modified siRNAs. Since 2018, four siRNA drugs - patisiran, givosiran, lumasiran, and inclisiran, were approved by the US FDA, providing a testament to the promise of RNAi therapeutics. Despite these promising results, safe and efficient siRNA delivery at the target site remains a major obstacle for efficient siRNA-based therapeutics. In this review, we have outlined the synergistic effects of emerging dual ribose modifications, including 2',4'- and 2',5'-modifications, 5'-E/Z-vinylphosphonate, and northern methanocarbacyclic (NMC) modifications that have contributed to drug-like effects in siRNA. These modifications enhance nuclease stability, prolong gene silencing efficiency, improve thermal stability, and exhibit high tissue accumulation. We also highlight the current progress in siRNA clinical trials. This review will help to understand the potential effects of dual ribose modifications and provides alternative ways to use extensive 2'-modifications in siRNA drugs. Moreover, the minimal number of these dual ribose modifications could be sufficient to achieve the desired therapeutic effect. In future, detailed in vivo studies using these dual ribose modifications could help to improve the therapeutic effects of siRNA. Rational design could further open doors for the rapid progress in siRNA therapeutics. [Figure: see text].

Folate Receptor-Mediated siRNA Delivery: Recent Developments and Future Directions for RNAi Therapeutics.

RNA interference (RNAi), a gene regulatory process mediated by small interfering RNAs (siRNAs), has made remarkable progress as a potential therapeutic agent against various diseases. However, RNAi is associated with fundamental challenges such as poor systemic delivery and susceptibility to the nucleases. Targeting ligand-bound delivery vehicles has improved the accumulation of drug at the target site, which has resulted in high transfection efficiency and enhanced gene silencing. Recently, folate receptor (FR)-mediated targeted delivery of siRNAs has garnered attention due to their enhanced cellular uptake and high transfection efficiency toward tumor cells. Folic acid (FA), due to its small size, low immunogenicity, high in vivo stability, and high binding affinity toward FRs, has attracted much attention for targeted siRNA delivery. FRs are overexpressed in a large number of tumors, including ovarian, breast, kidney, and lung cancer cells. In this review, we discuss recent advances in FA-mediated siRNA delivery to treat cancers and inflammatory diseases. This review summarizes various FA-conjugated nanoparticle systems reported so far in the literature, including liposome, silica, metal, graphene, dendrimers, chitosan, organic copolymers, and RNA nanoparticles. This review will help in the design and development of potential delivery vehicles for siRNA drug targeting to tumor cells using an FR-mediated approach.

A Multicolor Large Stokes Shift Fluorogen-Activating RNA Aptamer with Cationic Chromophores.

Large Stokes shift (LSS) fluorescent proteins (FPs) exploit excited state proton transfer pathways to enable fluorescence emission from the phenolate intermediate of their internal 4-hydroxybenzylidene imidazolone (HBI) chromophore. An RNA aptamer named Chili mimics LSS FPs by inducing highly Stokes-shifted emission from several new green and red HBI analogues that are non-fluorescent when free in solution. The ligands are bound by the RNA in their protonated phenol form and feature a cationic aromatic side chain for increased RNA affinity and reduced magnesium dependence. In combination with oxidative functionalization at the C2 position of the imidazolone, this strategy yielded DMHBO+ , which binds to the Chili aptamer with a low-nanomolar KD . Because of its highly red-shifted fluorescence emission at 592 nm, the Chili-DMHBO+ complex is an ideal fluorescence donor for Förster resonance energy transfer (FRET) to the rhodamine dye Atto 590 and will therefore find applications in FRET-based analytical RNA systems.

Journey of siRNA: Clinical Developments and Targeted Delivery.

Since the evolutionary discovery of RNA interference and its utilization for gene knockdown in mammalian cell, a remarkable progress has been achieved in small interfering RNA (siRNA) therapeutics. siRNA is a promising tool, utilized as therapeutic agent against various diseases. Despite its significant potential benefits, safe, efficient, and target oriented delivery of siRNA is one of the major challenges in siRNA therapeutics. This review covers major achievements in clinical trials and targeted delivery of siRNAs using various targeting ligand-receptor pair. Local and systemically administered siRNA drug candidates at various phases in clinical trials are described in this review. This review also provides a deep insight in development of targeted delivery of siRNA. Various targeting ligand-siRNA pair with complexation and conjugation approaches are discussed in this review. This will help to achieve further optimization and development in targeted delivery of siRNAs to achieve higher gene silencing efficiency with lowest siRNA dose availability.

The N(2)-Furfuryl-deoxyguanosine Adduct Does Not Alter the Structure of B-DNA.

N(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 N(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 E. coli. Introduction of fdG into a DNA duplex decreases the Tm 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.

Unique structural features in DNA polymerase IV enable efficient bypass of the N2 adduct induced by the nitrofurazone antibiotic.

The reduction in the efficacy of therapeutic antibiotics represents a global problem of increasing intensity and concern. Nitrofuran antibiotics act primarily through the formation of covalent adducts at the N(2) atom of the deoxyguanosine nucleotide in genomic DNA. These adducts inhibit replicative DNA polymerases (dPols), leading to the death of the prokaryote. N(2)-furfuryl-deoxyguanosine (fdG) represents a stable structural analog of the nitrofuran-induced adducts. Unlike other known dPols, DNA polymerase IV (PolIV) from E. coli can bypass the fdG adduct accurately with high catalytic efficiency. This property of PolIV is central to its role in reducing the sensitivity of E. coli toward nitrofuran antibiotics such as nitrofurazone (NFZ). We present the mechanism used by PolIV to bypass NFZ-induced adducts and thus improve viability of E. coli in the presence of NFZ. Our results can be used to develop specific inhibitors of PolIV that may potentiate the activity of nitrofuran antibiotics.

Influence of 2'-fluoro versus 2'-O-methyl substituent on the sugar puckering of 4'-C-aminomethyluridine.

Herein, we report the synthesis of 4'-C-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'-C-CH2 group and the F atom at the 2' position is observed. Comparative studies with unmodified, 2'-fluoro-, 2'-O-methyl-, and 4'-C-aminomethyl-2'-O-methyluridine showed the chemical nature of 2'-substituent dictates the sugar puckering of 2',4'-modified nucleotides.

Design, synthesis, biophysical and primer extension studies of novel acyclic butyl nucleic acid (BuNA).

A novel nucleic acid analogue called acyclic (S)-butyl nucleic acid (BuNA) composed of an acyclic backbone containing a phosphodiester linkage and bearing natural nucleobases was synthesized. Next, (S)-BuNA nucleotides were incorporated in DNA strands and their effect on duplex stability and changes in structural conformation were investigated. Circular dichroism (CD), UV-melting and non-denatured gel electrophoresis (native PAGE) studies revealed that (S)-BuNA is capable of making duplexes with its complementary strands and integration of (S)-BuNA nucleotides into DNA duplex does not alter the B-type-helical structure of the duplex. Furthermore, (S)-BuNA oligonucleotides and (S)-BuNA substituted DNA strands were studied as primer extensions by DNA polymerases. This study revealed that the acyclic scaffold is tolerated by enzymes and is therefore to some extent biocompatible.

Incorporation of 4'-C-aminomethyl-2'-O-methylthymidine into DNA by thermophilic DNA polymerases.

The dual modified nucleotide 4'-C-aminomethyl-2'-O-methylthymidine 5'-triphosphate was synthesized and enzymatically incorporated into DNA by the thermophilic DNA polymerases Pfu and Therminator III. The dual ribose modification imparted increased exonuclease resistance to DNA compared to the well-known 2'-O-methyl modification.

Synthesis, gene silencing, and molecular modeling studies of 4'-C-aminomethyl-2'-O-methyl modified small interfering RNAs.

The linear syntheses of 4'-C-aminomethyl-2'-O-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 T(m) (∼1 °C/modification) compared to the unmodified duplex. Molecular dynamics simulations revealed that the 4'-C-aminomethyl-2'-O-methyl modified nucleotides adopt South-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'-C-aminomethyl-2'-O-methyl modified nucleotides to overcome some of the challenges associated with the therapeutic utilities of siRNAs.