Prolinyl Phosphoramidates of Nucleotides with Increased Reactivity.

Nucleoside monophosphates (NMPs) are the subunits of RNA. They are incorporated into growing complementary strands when sequences are copied in enzyme-free reactions using organic leaving groups at the phosphates. Amino acids are rarely considered as leaving groups, but proline can act as a leaving group when N-linked to NMPs, so that prolinyl NMPs hydrolyze in aqueous buffer at 37 °C, with half-life times as short as 2.4 h, and they act as monomers in enzyme-free primer extension. Still, their level of reactivity is insufficient for practical purposes, requiring months for some extensions. Herein we report the synthesis of eight substituted prolinyl AMPs together with seven related compounds and the results of a study of their reactivity. A δ-carboxy prolinyl NMP was found to be converted with a half-life time of just 11 min in magnesium-free buffer, and a δ-isopropyl prolinyl NMP was shown to react sevenfold faster than its prolinyl counterpart in enzyme-free genetic copying of RNA. Our results indicate that both anchimeric and steric effects can be employed to increase the reactivity of aminoacidyl nucleotides, i.e. compounds that combine two fundamental classes of biomolecules in one functional entity.

Replication Bypass of the N-(2-Deoxy-d-erythro-pentofuranosyl)-urea DNA Lesion by Human DNA Polymerase η.

Urea lesions in DNA arise from thymine glycol (Tg) or 8-oxo-dG; their genotoxicity is thought to arise in part due to their potential to accommodate the insertion of all four dNTPs during error-prone replication. Replication bypass with human DNA polymerase η (hPol η) confirmed that all four dNTPs were inserted opposite urea lesions but with purines exhibiting greater incorporation efficiency. X-ray crystal structures of ternary replication bypass complexes in the presence of Mg2+ ions with incoming dNTP analogs dAMPnPP, dCMPnPP, dGMPnPP, and dTMPnPP bound opposite urea lesions (hPol η·DNA·dNMPnPP complexes) revealed all were accommodated by hPol η. In each, the Watson-Crick face of the dNMPnPP was paired with the urea lesion, exploiting the ability of the amine and carbonyl groups of the urea to act as H-bond donors or acceptors, respectively. With incoming dAMPnPP or dGMPnPP, the distance between the imino nitrogen of urea and the N9 atoms of incoming dNMPnPP approximated the canonical distance of 9 Å in B-DNA. With incoming dCMPnPP or dTMPnPP, the corresponding distance of about 7 Å was less ideal. Improved base-stacking interactions were also observed with incoming purines vs pyrimidines. Nevertheless, in each instance, the α-phosphate of incoming dNMPnPPs was close to the 3'-hydroxyl group of the primer terminus, consistent with the catalysis of nucleotidyl transfer and the observation that all four nucleotides could be inserted opposite urea lesions. Preferential insertion of purines by hPol η may explain, in part, why the urea-directed spectrum of mutations arising from Tg vs 8-oxo-dG lesions differs.

Synthesis and Biophysical Studies of High-Affinity Morpholino Oligomers Containing G-Clamp Analogs.

Successful syntheses of chlorophosphoramidate morpholino monomers containing tricyclic cytosine analogs phenoxazine, G-clamp, and G8AE-clamp were accomplished. These modified monomers were incorporated into 12-mer oligonucleotides using trityl-chemistry by an automated synthesizer. The resulting phosphorodiamidate morpholino oligomers, containing a single G-clamp, demonstrated notably higher affinity for complementary RNA and DNA compared to the unmodified oligomers under neutral and acidic conditions. The duplexes of RNA and DNA with G-clamp-modified oligomers adopt a B-type helical conformation, as evidenced by CD-spectra and show excellent base recognition properties. Binding affinities were sequence and position dependent.

Robert Letsinger and the Evolution of Oligonucleotide Synthesis.

This retrospective summarizes a presentation given at the symposium "Bob Letsinger, PhD-100 Years of History" on September 28, 2021 as part of the 17th annual meeting of the Oligonucleotide Therapeutics Society (OTS). In it I look back at my encounters with Robert Lewis Letsinger (1921-2014) while at Northwestern University as Assistant Professor between 1995 and 2000.

Conformational Morphing by a DNA Analogue Featuring 7-Deazapurines and 5-Halogenpyrimidines and the Origins of Adenine-Tract Geometry.

Several efforts are currently directed at the creation and cellular implementation of alternative genetic systems composed of pairing components that are orthogonal to the natural dA/dT and dG/dC base pairs. In an alternative approach, Watson-Crick-type pairing is conserved, but one or all of the four letters of the A, C, G, and T alphabet are substituted by modified components. Thus, all four nucleobases were altered to create halogenated deazanucleic acid (DZA): dA was replaced by 7-deaza-2'-deoxyadenosine (dzA), dG by 7-deaza-2'-deoxyguanosine (dzG), dC by 5-fluoro-2'-deoxycytidine (FdC), and dT by 5-chloro-2'-deoxyuridine (CldU). This base-pairing system was previously shown to retain function in Escherichia coli. Here, we analyze the stability, hydration, structure, and dynamics of a DZA Dickerson-Drew Dodecamer (DDD) of sequence 5'-FdC-dzG-FdC-dzG-dzA-dzA-CldU-CldU-FdC-dzG-FdC-dzG-3'. Contrary to similar stabilities of DDD and DZA-DDD, osmotic stressing revealed a dramatic loss of hydration for the DZA-DDD relative to that for the DDD. The parent DDD 5'-d(CGCGAATTCGCG)-3' features an A-tract, a run of adenosines uninterrupted by a TpA step, and exhibits a hallmark narrow minor groove. Crystal structures─in the presence of RNase H─and MD simulations show increased conformational plasticity ("morphing") of DZA-DDD relative to that of the DDD. The narrow dzA-tract minor groove in one structure widens to resemble that in canonical B-DNA in a second structure. These changes reflect an indirect consequence of altered DZA major groove electrostatics (less negatively polarized compared to that in DNA) and hydration (reduced compared to that in DNA). Therefore, chemical modifications outside the minor groove that lead to collapse of major groove electrostatics and hydration can modulate A-tract geometry.

Shorter Is Better: The α-(l)-Threofuranosyl Nucleic Acid Modification Improves Stability, Potency, Safety, and Ago2 Binding and Mitigates Off-Target Effects of Small Interfering RNAs.

Chemical modifications are necessary to ensure the metabolic stability and efficacy of oligonucleotide-based therapeutics. Here, we describe analyses of the α-(l)-threofuranosyl nucleic acid (TNA) modification, which has a shorter 3'-2' internucleotide linkage than the natural DNA and RNA, in the context of small interfering RNAs (siRNAs). The TNA modification enhanced nuclease resistance more than 2'-O-methyl or 2'-fluoro ribose modifications. TNA-containing siRNAs were prepared as triantennary N-acetylgalactosamine conjugates and were tested in cultured cells and mice. With the exceptions of position 2 of the antisense strand and position 11 of the sense strand, the TNA modification did not inhibit the activity of the RNA interference machinery. In a rat toxicology study, TNA placed at position 7 of the antisense strand of the siRNA mitigated off-target effects, likely due to the decrease in the thermodynamic binding affinity relative to the 2'-O-methyl residue. Analysis of the crystal structure of an RNA octamer with a single TNA on each strand showed that the tetrose sugar adopts a C4'-exo pucker. Computational models of siRNA antisense strands containing TNA bound to Argonaute 2 suggest that TNA is well accommodated in the region kinked by the enzyme. The combined data indicate that the TNA nucleotides are promising modifications expected to increase the potency, duration of action, and safety of siRNAs.

The peroxidation-derived DNA adduct, 6-oxo-M1dG, is a strong block to replication by human DNA polymerase η.

The DNA adduct 6-oxo-M1dG, (3-(2'-deoxy-ß-D-erythro-pentofuranosyl)-6-oxo-pyrimido(1,2alpha)purin-10(3H)-one) is formed in the genome via oxidation of the peroxidation-derived adduct M1dG. However, the effect of 6-oxo-M1dG adducts on subsequent DNA replication is unclear. Here we investigated the ability of the human Y-family polymerase hPol η to bypass 6-oxo-M1dG. Using steady-state kinetics and analysis of DNA extension products by liquid chromatography-tandem mass spectrometry, we found hPol η preferentially inserts a dAMP or dGMP nucleotide into primer-templates across from the 6-oxo-M1dG adduct, with dGMP being slightly preferred. We also show primer-templates with a 3'-terminal dGMP or dAMP across from 6-oxo-M1dG were extended to a greater degree than primers with a dCMP or dTMP across from the adduct. In addition, we explored the structural basis for bypass of 6-oxo-M1dG by hPol η using X-ray crystallography of both an insertion-stage and an extension-stage complex. In the insertion-stage complex, we observed that the incoming dCTP opposite 6-oxo-M1dG, although present during crystallization, was not present in the active site. We found the adduct does not interact with residues in the hPol η active site but rather forms stacking interactions with the base pair immediately 3' to the adduct. In the extension-stage complex, we observed the 3' hydroxyl group of the primer strand dGMP across from 6-oxo-M1dG is not positioned correctly to form a phosphodiester bond with the incoming dCTP. Taken together, these results indicate 6-oxo-M1dG forms a strong block to DNA replication by hPol η and provide a structural basis for its blocking ability.

Rational optimization of siRNA to ensure strand bias in the interaction with the RNA-induced silencing complex.

To ensure specificity of small interfering RNAs (siRNAs), the antisense strand must be selected by the RNA-induced silencing complex (RISC). We have previously demonstrated that a 5'-morpholino-modified nucleotide at the 5'-end of the sense strand inhibits its interaction with RISC ensuring selection of the desired antisense strand. To improve this antagonizing binding property even further, a new set of morpholino-based analogues, Mo2 and Mo3, and a piperidine analogue, Pip, were designed based on the known structure of Argonaute2, the slicer enzyme component of RISC. Sense strands of siRNAs were modified with these new analogues, and the siRNAs were evaluated in vitro and in mice for RNAi activity. Our data demonstrated that Mo2 is the best RISC inhibitor among the modifications tested and that it effectively mitigates sense strand-based off-target activity of siRNA.

Base excision repair of the N-(2-deoxy-d-erythro-pentofuranosyl)-urea lesion by the hNEIL1 glycosylase.

The N-(2-deoxy-d-erythro-pentofuranosyl)-urea DNA lesion forms following hydrolytic fragmentation of cis-5R,6S- and trans-5R,6R-dihydroxy-5,6-dihydrothymidine (thymine glycol, Tg) or from oxidation of 7,8-dihydro-8-oxo-deoxyguanosine (8-oxodG) and subsequent hydrolysis. It interconverts between α and ß deoxyribose anomers. Synthetic oligodeoxynucleotides containing this adduct are efficiently incised by unedited (K242) and edited (R242) forms of the hNEIL1 glycosylase. The structure of a complex between the active site unedited mutant CΔ100 P2G hNEIL1 (K242) glycosylase and double-stranded (ds) DNA containing a urea lesion reveals a pre-cleavage intermediate, in which the Gly2 N-terminal amine forms a conjugate with the deoxyribose C1' of the lesion, with the urea moiety remaining intact. This structure supports a proposed catalytic mechanism in which Glu3-mediated protonation of O4' facilitates attack at deoxyribose C1'. The deoxyribose is in the ring-opened configuration with the O4' oxygen protonated. The electron density of Lys242 suggests the 'residue 242-in conformation' associated with catalysis. This complex likely arises because the proton transfer steps involving Glu6 and Lys242 are hindered due to Glu6-mediated H-bonding with the Gly2 and the urea lesion. Consistent with crystallographic data, biochemical analyses show that the CΔ100 P2G hNEIL1 (K242) glycosylase exhibits a residual activity against urea-containing dsDNA.

Chemistry, structure and function of approved oligonucleotide therapeutics.

Eighteen nucleic acid therapeutics have been approved for treatment of various diseases in the last 25 years. Their modes of action include antisense oligonucleotides (ASOs), splice-switching oligonucleotides (SSOs), RNA interference (RNAi) and an RNA aptamer against a protein. Among the diseases targeted by this new class of drugs are homozygous familial hypercholesterolemia, spinal muscular atrophy, Duchenne muscular dystrophy, hereditary transthyretin-mediated amyloidosis, familial chylomicronemia syndrome, acute hepatic porphyria, and primary hyperoxaluria. Chemical modification of DNA and RNA was central to making drugs out of oligonucleotides. Oligonucleotide therapeutics brought to market thus far contain just a handful of first- and second-generation modifications, among them 2'-fluoro-RNA, 2'-O-methyl RNA and the phosphorothioates that were introduced over 50 years ago. Two other privileged chemistries are 2'-O-(2-methoxyethyl)-RNA (MOE) and the phosphorodiamidate morpholinos (PMO). Given their importance in imparting oligonucleotides with high target affinity, metabolic stability and favorable pharmacokinetic and -dynamic properties, this article provides a review of these chemistries and their use in nucleic acid therapeutics. Breakthroughs in lipid formulation and GalNAc conjugation of modified oligonucleotides have paved the way to efficient delivery and robust, long-lasting silencing of genes. This review provides an account of the state-of-the-art of targeted oligo delivery to hepatocytes.

Synthesis and Biophysical Properties of Phosphorodiamidate Piperidino Oligomers.

We report the synthesis of piperidino nucleoside phosphoramidates functionalized with uracil, cytosine, guanine, and adenine and their incorporation into oligomers. High-performance liquid chromatography analyses demonstrated that a phosphorodiamidate piperidino oligomer (PPO) is more lipophilic than a phosphorodiamidate morpholino oligomer (PMO) of the same tetrameric sequence. A PMO containing piperidino residues formed duplexes with both DNA and RNA, and the PPO had higher stability at endosomolytic pH and higher hydrophobicity than the PMO.

Acyclic (S)-glycol nucleic acid (S-GNA) modification of siRNAs improves the safety of RNAi therapeutics while maintaining potency.

Glycol nucleic acid (GNA) is an acyclic nucleic acid analog connected via phosphodiester bonds. Crystal structures of RNA-GNA chimeric duplexes indicated that nucleotides of the right-handed (S)-GNA were better accommodated in the right-handed RNA duplex than were the left-handed (R)-isomers. GNA nucleotides adopt a rotated nucleobase orientation within all duplex contexts, pairing with complementary RNA in a reverse Watson-Crick mode, which explains the inabilities of GNA C and G to form strong base pairs with complementary nucleotides. Transposition of the hydrogen bond donor and acceptor pairs using novel (S)-GNA isocytidine and isoguanosine nucleotides resulted in stable base-pairing with the complementary G and C ribonucleotides, respectively. GNA nucleotide or dinucleotide incorporation into an oligonucleotide increased resistance against 3'-exonuclease-mediated degradation. Consistent with the structural observations, small interfering RNAs (siRNAs) modified with (S)-GNA had greater in vitro potencies than identical sequences containing (R)-GNA. (S)-GNA is well tolerated in the seed regions of antisense and sense strands of a GalNAc-conjugated siRNA in vitro. The siRNAs containing a GNA base pair in the seed region had in vivo potency when subcutaneously injected into mice. Importantly, seed pairing destabilization resulting from a single GNA nucleotide at position 7 of the antisense strand mitigated RNAi-mediated off-target effects in a rodent model. Two GNA-modified siRNAs have shown an improved safety profile in humans compared with their non-GNA-modified counterparts, and several additional siRNAs containing the GNA modification are currently in clinical development.

Metabolically Stable Anomeric Linkages Containing GalNAc-siRNA Conjugates: An Interplay among ASGPR, Glycosidase, and RISC Pathways.

Conjugation of synthetic triantennary N-acetyl-d-galactosamine (GalNAc) to small interfering RNA (siRNA) mediates binding to the asialoglycoprotein receptor (ASGPR) on the surface of hepatocytes, facilitating liver-specific uptake and siRNA-mediated gene silencing. The natural ß-glycosidic bond of the GalNAc ligand is rapidly cleaved by glycosidases in vivo. Novel GalNAc ligands with S-, and C-glycosides with both α- and ß-anomeric linkages, N-glycosides with ß-anomeric linkage, and the O-glycoside with α-anomeric linkage were synthesized and conjugated to siRNA either on-column during siRNA synthesis or through a high-throughput, post-synthetic method. Unlike natural GalNAc, modified ligands were resistant to glycosidase activity. The siRNAs conjugated to newly designed ligands had similar affinities for ASGPR and similar silencing activity in mice as the parent GalNAc-siRNA conjugate. These data suggest that other factors, such as protein-nucleic acid interactions and loading of the antisense strand into the RNA-induced silencing complex (RISC), are more critical to the duration of action than the stereochemistry and stability of the anomeric linkage between the GalNAc moiety of the ligand conjugated to the sense strand of the siRNA.

Conformation and Pairing Properties of an O6-Methyl-2'-deoxyguanosine-Directed Benzimidazole Nucleoside Analog in Duplex DNA.

O6-Methyl-2'-deoxyguanosine (O6-MeG) is one of the most common DNA lesions and arises as a consequence of both xenobiotic carcinogens and endogenous methylation by S-adenosylmethionine. O6-MeG frequently causes G-to-A mutations during DNA replication due to the misincorporation of dTTP and continued DNA synthesis. Efforts to detect DNA adducts such as O6-MeG, and to understand their impacts on DNA structure and function, have motivated the creation of nucleoside analogs with altered base moieties to afford a more favorable interaction with the adduct as compared to the unmodified nucleotide. Such analogs directed at O6-MeG include benzimidazolinone and benzimidazole nucleotides, as well as their extended π surface analogs naphthimidazolinone and napthimidazole derivatives. These analogs form a more stable pair with O6-MeG than with G, most likely due to a combination of H-bonding and stacking. While extending the π surface of the analogs enhances their performance as adduct-directed probes, the precise origins of the increased affinity between the synthetic analogs and O6-MeG remain unclear. To better understand relevant conformational and pairing properties, we used X-ray crystallography and analyzed the structures of the DNA duplexes with naphthimidazolinone inserted opposite G or O6-MeG. The structures reveal a complex interaction of the analog found either in an anti orientation and stacked inside the duplex, either above or below G or O6-MeG, or in a syn orientation and paired opposite G with formation of a single H-bond. The experimental structural data are consistent with the stabilizing effect of the synthetic analog observed in UV melting experiments and calculations and moreover reveal that the origin of these observations appears to be superior stacking between O6-MeG and the extended π system of the synthetic probe.

A Chemical Approach to Introduce 2,6-Diaminopurine and 2-Aminoadenine Conjugates into Oligonucleotides without Need for Protecting Groups.

We report a simple, postsynthetic strategy for synthesis of oligonucleotides containing 2,6-diaminopurine nucleotides and 2-aminoadenine conjugates using 2-fluoro-6-amino-adenosine. The strategy allows introduction of 2,6-diaminopurine and other 2-amino group-containing ligands. The strongly electronegative 2-fluoro deactivates 6-NH2 obviating the need for any protecting group on adenine, and simple aromatic nucleophilic substitution of fluorine makes reaction with aqueous NH3 or R-NH2 feasible at the 2-position.

Role of a "Magic" Methyl: 2'-Deoxy-2'-α-F-2'-ß-C-methyl Pyrimidine Nucleotides Modulate RNA Interference Activity through Synergy with 5'-Phosphate Mimics and Mitigation of Off-Target Effects.

Although 2'-deoxy-2'-α-F-2'-ß-C-methyl (2'-F/Me) uridine nucleoside derivatives are a successful class of antiviral drugs, this modification had not been studied in oligonucleotides. Herein, we demonstrate the facile synthesis of 2'-F/Me-modified pyrimidine phosphoramidites and their subsequent incorporation into oligonucleotides. Despite the C3'-endo preorganization of the parent nucleoside, a single incorporation into RNA or DNA resulted in significant thermal destabilization of a duplex due to unfavorable enthalpy, likely resulting from steric effects. When located at the terminus of an oligonucleotide, the 2'-F/Me modification imparted more resistance to degradation than the corresponding 2'-fluoro nucleotides. Small interfering RNAs (siRNAs) modified at certain positions with 2'-F/Me had similar or better silencing activity than the parent siRNAs when delivered via a lipid nanoparticle formulation or as a triantennary N-acetylgalactosamine conjugate in cells and in mice. Modification in the seed region of the antisense strand at position 6 or 7 resulted in an activity equivalent to the parent in mice. Additionally, placement of the antisense strand at position 7 mitigated seed-based off-target effects in cell-based assays. When the 2'-F/Me modification was combined with 5'-vinyl phosphonate, both E and Z isomers had silencing activity comparable to the parent. In combination with other 2'-modifications such as 2'-O-methyl, the Z isomer is detrimental to silencing activity. Presumably, the equivalence of 5'-vinyl phosphonate isomers in the context of 2'-F/Me is driven by the steric and conformational features of the C-methyl-containing sugar ring. These data indicate that 2'-F/Me nucleotides are promising tools for nucleic acid-based therapeutic applications to increase potency, duration, and safety.

EWALD: A macromolecular diffractometer for the second target station.

Revealing the positions of all the atoms in large macromolecules is powerful but only possible with neutron macromolecular crystallography (NMC). Neutrons provide a sensitive and gentle probe for the direct detection of protonation states at near-physiological temperatures and clean of artifacts caused by x rays or electrons. Currently, NMC use is restricted by the requirement for large crystal volumes even at state-of-the-art instruments such as the macromolecular neutron diffractometer at the Spallation Neutron Source. EWALD's design will break the crystal volume barrier and, thus, open the door for new types of experiments, the study of grand challenge systems, and the more routine use of NMC in biology. EWALD is a single crystal diffractometer capable of collecting data from macromolecular crystals on orders of magnitude smaller than what is currently feasible. The construction of EWALD at the Second Target Station will cause a revolution in NMC by enabling key discoveries in the biological, biomedical, and bioenergy sciences.

Cryo neutron crystallography demonstrates influence of RNA 2'-OH orientation on conformation, sugar pucker and water structure.

The ribose 2'-hydroxyl is the key chemical difference between RNA and DNA and primary source of their divergent structural and functional characteristics. Macromolecular X-ray diffraction experiments typically do not reveal the positions of hydrogen atoms. Thus, standard crystallography cannot determine 2'-OH orientation (H2'-C2'-O2'-HO2' torsion angle) and its potential roles in sculpting the RNA backbone and the expansive fold space. Here, we report the first neutron crystal structure of an RNA, the Escherichia coli rRNA Sarcin-Ricin Loop (SRL). 2'-OD orientations were established for all 27 residues and revealed O-D bonds pointing toward backbone (O3', 13 observations), nucleobase (11) or sugar (3). Most riboses in the SRL stem region show a 2'-OD backbone-orientation. GAGA-tetraloop riboses display a 2'-OD base-orientation. An atypical C2'-endo sugar pucker is strictly correlated with a 2'-OD sugar-orientation. Neutrons reveal the strong preference of the 2'-OH to donate in H-bonds and that 2'-OH orientation affects both backbone geometry and ribose pucker. We discuss 2'-OH and water molecule orientations in the SRL neutron structure and compare with results from a solution phase 10 µs MD simulation. We demonstrate that joint cryo-neutron/X-ray crystallography offers an all-in-one approach to determine the complete structural properties of RNA, i.e. geometry, conformation, protonation state and hydration structure.

From bench to bedside: Improving the clinical safety of GalNAc-siRNA conjugates using seed-pairing destabilization.

Preclinical mechanistic studies have pointed towards RNA interference-mediated off-target effects as a major driver of hepatotoxicity for GalNAc-siRNA conjugates. Here, we demonstrate that a single glycol nucleic acid or 2'-5'-RNA modification can substantially reduce small interfering RNA (siRNA) seed-mediated binding to off-target transcripts while maintaining on-target activity. In siRNAs with established hepatotoxicity driven by off-target effects, these novel designs with seed-pairing destabilization, termed enhanced stabilization chemistry plus (ESC+), demonstrated a substantially improved therapeutic window in rats. In contrast, siRNAs thermally destabilized to a similar extent by the incorporation of multiple DNA nucleotides in the seed region showed little to no improvement in rat safety suggesting that factors in addition to global thermodynamics play a role in off-target mitigation. We utilized the ESC+ strategy to improve the safety of ALN-HBV, which exhibited dose-dependent, transient and asymptomatic alanine aminotransferase elevations in healthy volunteers. The redesigned ALN-HBV02 (VIR-2218) showed improved specificity with comparable on-target activity and the program was reintroduced into clinical development.