Rajat S. Das

The conjugation of the sense strands of small interfering RNA (siRNA) to tri-N-acetylgalactosamine (GalNAc), the ligand for a hepatocyte-specific receptor, enables the delivery of multiple clinically approved therapeutic agents that act through the RNA interference pathway. Here, we report the systematic evaluation of siRNAs with the 3' termini of antisense strands conjugated to GalNAc for the first time. These designs retained the same receptor affinity, in vitro and in vivo activities, as well as the same level of loading into the RNA-induced silencing complex as siRNAs with a GalNAc-conjugated sense strand. A siRNA with a GalNAc-conjugated antisense strand of 22 nucleotides had better activity than a siRNA with a 23-nucleotide antisense strand. Computational modeling of a complex of a GalNAc-conjugated antisense strand with the PAZ domain of Ago2 rationalizes the importance of the interaction of phosphate at the 3' terminus with the PAZ domain to explain the observed activity of these siRNAs.

2′-Fluorinated Northern methanocarbacyclic (2′-F-NMC) nucleosides and phosphoramidites, based on a bicyclo[3.1.0]hexane scaffold bearing all four natural nucleobases (U, C, A, and G), were synthesized to enable exploration of this novel nucleotide modification related to the clinically validated 2′-deoxy-2′-fluororibonucleotides (2′-F-RNA). Biophysical properties of the 2′-F-NMC-containing oligonucleotides were evaluated. A duplex of 2′-F-NMC-modified oligonucleotide with RNA exhibited thermal stability similar to that of the parent RNA duplex, 2′-F-NMC-modified oligonucleotides had higher stability against 5′- and 3′-exonucleolytic degradation than the corresponding oligonucleotides modified with 2′-F-RNA, and 2′-F-NMC-modified oligonucleotides exhibited higher lipophilicity than the corresponding RNA oligonucleotides as well as those modified with 2′-F-RNA.

Middle East respiratory syndrome coronavirus (MERS-CoV) remains a threat to public health worldwide; however, effective vaccine or drug against CoVs remains unavailable. CoV helicase is one of the three evolutionary most conserved proteins in nidoviruses, thus making it an important target for drug development. We report here the first structure of full-length coronavirus helicase, MERS-CoV nsp13. MERS-CoV helicase has multiple domains, including an N-terminal Cys/His rich domain (CH) with three zinc atoms, a beta-barrel domain and a C-terminal SF1 helicase core with two RecA-like subdomains. Our structural analyses show that while the domain organization of nsp13 is conserved throughout nidoviruses, the individual domains of nsp13 are closely related to the equivalent eukaryotic domains of Upf1 helicases. The most distinctive feature differentiating CoV helicases from eukaryotic Upf1 helicases is the interaction between CH domain and helicase core. Severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) caused global pandemics in 2003 and 2012 with the fatality rates of 10–35%. Outbreak of MERS-CoV in the Republic of Korea in 2015 highlighted that the newly emerged CoVs remain a concern for the public health. Nevertheless, effective vaccine and drug against CoVs are still missing. Among CoV-encoded nonstructural proteins (nsps), nsp13 helicase is considered one of the most important drug targets. Nsp13 is a highly conserved protein in CoVs and nidovirales and one of the two central components of the membrane associated replication-transcription complex, which performs viral RNA synthesis. However, despite decades of structural characterization of CoV-encoded proteins, the structure of nsp13 remained unavailable. In this study, we determined the first crystal structure of the full-length MERS-CoV nsp13. MERS-CoV helicase has an N-terminal Cys/His rich domain (CH) with three zincs, a beta-barrel domain and a C-terminal SF1 helicase core. While the domain organization of nsp13 is similar to arterivirus nsp10, the individual domains of nsp13 are closely related to their equivalent domains of eukaryotic Upf1 helicases. Our findings provide novel structural information essential for structure-based drug design against CoV.

Displacing the hydroxyl nucleophile at the 3′-end of a primer by a single methylene group drastically decreases the rate of primer extension, illustrating the importance of the precise position of the hydroxyl nucleophile.

To facilitate NMR studies and low‐level detection in biological samples by mass spectrometry, [1,3, NH₂‐¹⁵N₃] (5′S)‐8,5′‐cyclo‐2′‐deoxyguanosine was synthesized from imidazole‐4,5‐dicarboxylic acid in 21 steps. The three ¹⁵N isotopes were introduced during the chemo‐enzymatic preparation of [1,3, NH₂‐¹⁵N₃]‐2′‐deoxyguanosine using an established procedure. The ¹⁵N‐labeled 2′‐deoxyguanosine was converted to a 5′‐phenylthio derivative, which allowed the 8‐5′ covalent bond formation via photochemical homolytic cleavage of the C–SPh bond. SeO₂ oxidation of C‐5′ followed by sodium borohydride reduction and deprotection gave the desired product in good yield. The isotopic purity of the [1,3, NH₂‐¹⁵N₃] (5′S)‐8,5′‐cyclo‐2′‐deoxyguanosine was in excess of 99.94 atom% based on liquid chromatography–mass spectrometry measurements. Copyright © 2013 John Wiley & Sons, Ltd.

8,5′-Cyclopurine deoxynucleosides are unique tandem lesions containing an additional covalent bond between the base and the sugar. These mutagenic and genotoxic lesions are repaired only by nucleotide excision repair. The N-glycosidic (or C1′-N9) bond of 2′-deoxyguanosine (dG) derivatives is usually susceptible to acid hydrolysis, but even after cleavage of this bond of the cyclopurine lesions, the base would remain attached to the sugar. Here, the stability of the N-glycosidic bond and the products formed by formic acid hydrolysis of (5′S)-8,5′-cyclo-2′-deoxyguanosine (S-cdG) were investigated. For comparison, the stability of the N-glycosidic bond of 8,5′-cyclo-2′,5′-dideoxyguanosine (ddcdG), 8-methyl-2′-deoxyguanosine (8-Me-dG), 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-Oxo-dG), and dG was also studied. In various acid conditions, S-cdG and ddcdG exhibited similar stability to hydrolysis. Likewise, 8-Me-dG and dG showed comparable stability, but the half-lives of the cyclic dG lesions were at least 5-fold higher than those of dG or 8-Me-dG. NMR studies were carried out to investigate the products formed after the cleavage of the C1′-N9 bond. 2-Deoxyribose generated α and β anomers of deoxyribopyranose and deoxyribopyranose oligomers following acid treatment. S-cdG gave α- and β-deoxyribopyranose linked guanine as the major products, but α and β anomers of deoxyribofuranose linked guanine and other products were also detected. The N-glycosidic bond of 8-Oxo-dG was found exceptionally stable in acid. Computational studies determined that both the protonation of the N7 atom and the rate constant in the bond breaking step control the overall kinetics of hydrolysis, but both varied for the molecules studied indicating a delicate balance between the two steps. Nevertheless, the computational approach successfully predicted the trend observed experimentally. For 8-Oxo-dG, the low pK_a of O⁸ and N3 prevented appreciable protonation, making the free energy for N-glycosidic bond cleavage in the subsequent step very high.

Diastereomeric 8,5′-cyclopurine 2′-deoxynucleosides, containing a covalent bond between the deoxyribose and the purine base, are induced in DNA by ionizing radiation. They are suspected to play a role in the etiology of neurodegeneration in xeroderma pigmentosum patients. If not repaired, the S-8,5′-cyclo-2′-deoxyguanosine lesion (S-cdG) induces Pol V-dependent mutations at a frequency of 34% in Escherichia coli. Most are S-cdG → A transitions, suggesting mis-incorporation of dTTP opposite the lesion during replication bypass, although low levels of S-cdG → T transversions, arising from mis-incorporation of dATP, are also observed. We report the structures of 5′-d(GTGCXTGTTTGT)-3′·5′-d(ACAAACAYGCAC)-3′, where X denotes S-cdG and Y denotes either dA or dT, corresponding to the situation following mis-insertion of either dTTP or dATP opposite the S-cdG lesion. The S-cdG·dT mismatch pair adopts a wobble base pairing. This provides a plausible rationale for the S-cdG → A transitions. The S-cdG·dA mismatch pair differs in conformation from the dG·dA mismatch pair. For the S-cdG·dA mismatch pair, both S-cdG and dA intercalate, but no hydrogen bonding is observed between S-cdG and dA. This is consistent with the lower levels of S-cdG → T transitions in E. coli.

Diastereomeric 8,5′-cyclopurine 2′-deoxynucleosides, containing a covalent bond between the deoxyribose and the purine base, represent an important class of DNA damage induced by ionizing radiation. The 8,5′-cyclo-2′-deoxyguanosine lesion (cdG) has been recently reported to be a strong block of replication and highly mutagenic in Escherichia coli. The 8,5′-cyclopurine-2′-deoxyriboses are suspected to play a role in the etiology of neurodegeneration in xeroderma pigmentosum patients. These lesions cannot be repaired by base excision repair, but they are substrates for nucleotide excision repair. The structure of an oligodeoxynucleotide duplex containing a site-specific S-cdG lesion placed opposite dC in the complementary strand was obtained by molecular dynamics calculations restrained by distance and dihedral angle restraints obtained from NMR spectroscopy. The S-cdG deoxyribose exhibited the O4′-exo (west) pseudorotation. Significant perturbations were observed for the β, γ, and χ torsion angles of the S-cdG nucleoside. Watson–Crick base pairing was conserved at the S-cdG·dC pair. However, the O4′-exo pseudorotation of the S-cdG deoxyribose perturbed the helical twist and base pair stacking at the lesion site and the 5′-neighbor dC·dG base pair. Thermodynamic destabilization of the duplex measured by UV melting experiments correlated with base stacking and structural perturbations involving the modified S-cdG·dC and 3′- neighbor dT·dA base pairs. These perturbations may be responsible for both the genotoxicity of this lesion and its ability to be recognized by nucleotide excision repair.

8,5′-Cyclopurines, making up an important class of ionizing radiation-induced tandem DNA damage, are repaired only by nucleotide excision repair (NER). They accumulate in NER-impaired cells, as in Cockayne syndrome group B and certain Xeroderma Pigmentosum patients. A plasmid containing (5′S)-8,5′-cyclo-2′-deoxyguanosine (S-cdG) was replicated in Escherichia coli with specific DNA polymerase knockouts. Viability was S-cdA > S-cdG. In summary, S-cdG is a major block to DNA replication, highly mutagenic, and repaired slowly in E. coli.