Sp Diastereomers Research Articles

Harnessing sulfur-binding domains to separate Sp and Rp isomers of phosphorothioate oligonucleotides

Chemical synthesis of phosphoromonothioate oligonucleotides (PS-ONs) is not stereo-specific and produces a mixture of Rp and Sp diastereomers, whose disparate reactivity can complicate applications. Although the current methods to separate these diastereomers which rely on chromatography are constantly improving, many Rp and Sp diastereomers are still co-eluted. Here, based on sulfur-binding domains that specifically recognize phosphorothioated DNA and RNA in Rp configuration, we developed a universal separation system for phosphorothioate oligonucleotide isomers using immobilized SBD (SPOIS). With the scalable SPOIS, His-tagged SBD is immobilized onto Ni-nitrilotriacetic acid-coated magnetic beads to form a beads/SBD complex, Rp isomers of the mixture can be completely bound by SBD and separated from Sp isomers unbound in liquid phase, then recovered through suitable elution approach. Using the phosphoromonothioate single-stranded DNA as a model, SPOIS separated PS-ON diastereomers of 4 nt to 50 nt in length at yields of 60–90% of the starting Rp isomers, with PS linkage not locating at 5’ or 3’ end. Within this length range, PS-ON diastereomers that co-eluted in HPLC could be separated by SPOIS at yields of 84% and 89% for Rp and Sp stereoisomers, respectively. Furthermore, as each Rp phosphorothioate linkage can be bound by SBD, SPOIS allowed the separation of stereoisomers with multiple uniform Sp configurations for multiple phosphorothioate modifications. A second generation of SPOIS was developed using the thermolabile and non-sequence-specific SBDPed, enabling fast and high-yield recovery of PS substrate stereoisomers for the DNAzyme Cd16 and further demonstrating the efficiency of this method.Key points• SPOIS allows isomer separations of the Rp and Sp isomers co-eluted on HPLC.• SPOIS can obtain Sp isomers with 5 min and Rp in 20 min from PS-ON diastereomers.• SPOIS was successfully applied to separate isomers of PS substrates of DNAzyme.Graphical

Double-modified, thio and methylene ATP analogue facilitates wound healing in vitro and in vivo

Recent data indicate that extracellular ATP affects wound healing efficacy via P2Y2-dependent signaling pathway. In the current work, we propose double-modified ATP analogue—alpha-thio-beta,gamma-methylene-ATP as a potential therapeutic agent for a skin regeneration. For the better understanding of structure–activity relationship, beside tested ATP analogues, the appropriate single-modified derivatives of target compound, such as alpha-thio-ATP and beta,gamma-methylene-ATP, were also tested in the context of their involvement in the activation of ATP-dependent purinergic signaling pathway via the P2Y2 receptor. The diastereomerically pure alpha-thio-modified-ATP derivatives were obtained using the oxathiaphospholane method as separate SP and RP diastereomers. Both the single- and double- modified ATP analogues were then tested for their impact on the viability and migration of human keratinocytes. The involvement of P2Y2-dependent purinergic signaling was analyzed in silico by molecular docking of the tested compounds to the P2Y2 receptor and experimentally by studying intracellular calcium mobilization in the human keratinocytes HaCaT. The effects obtained for ATP analogues were compared with the results for ATP as a natural P2Y2 agonist. To confirm the contribution of the P2Y2 receptor to the observed effects, the tests were also performed in the presence of the selective P2Y2 antagonist—AR-C118925XX. The ability of the alpha-thio-beta,gamma-methylene-ATP to influence cell migration was analyzed in vitro on the model HaCaT and MDA-MB-231 cells by wound healing assay and transwell migration test as well as in vivo using zebrafish system. The impact on tissue regeneration was estimated based on the regrowth rate of cut zebrafish tails. The in vitro and in vivo studies have shown that the SP-alpha-thio-beta,gamma-methylene-ATP analogue promotes regeneration-related processes, making it a suitable agent for enhance wound healing. Performed studies indicated its impact on the cell migration, induction of epithelial–mesenchymal transition and intracellular calcium mobilization. The enhanced regeneration of cut zebrafish tails confirmed the pro-regenerative activity of this ATP analogue. Based on the performed studies, the SP-alpha-thio-beta,gamma-methylene-ATP is proposed as a potential therapeutic agent for wound healing and skin regeneration treatment.

Replication Studies of Alkyl Phosphotriester Lesions in Human Cells.

Alkyl phosphotriester (alkyl-PTE) lesions in DNA are shown to be poorly repaired; however, little is known about how these lesions impact DNA replication in human cells. Here, we investigated how the SP and RP diastereomers of four alkyl-PTE lesions (alkyl = Me, Et, nPr, or nBu) at the TT site perturb DNA replication in HEK293T cells. We found that these lesions moderately impede DNA replication and that their replicative bypass is accurate. Moreover, CRISPR-Cas9-mediated depletion of Pol η or Pol ζ resulted in significantly attenuated bypass efficiencies for both diastereomers of nPr- and nBu-PTE adducts, and the SP diastereomer of Et-PTE. Diminished bypass efficiencies were also detected for the Rp diastereomer of nPr- and nBu-PTE lesions upon ablation of Pol κ. Together, our study uncovered the impact of the alkyl-PTE lesions on DNA replication in human cells and revealed the roles of individual translesion synthesis DNA polymerases in bypassing these lesions.

The Influence of Spirodi(Iminohydantoin) on Charge Transfer through ds-DNA Containing 8-OXO-dG: A Theoretical Approach.

Genetic information stored in a DNA base sequence is continuously exposed to harmful factors. It has been determined that 9 × 104 different DNA damage events occur in a single human cell every 24 h. Of these, 7,8-dihydro-8-oxo-guanosine (OXOG) is one of the most abundant and can undergo further transformations towards spirodi(iminohydantoin) (Sp). Sp is highly mutagenic in comparison to its precursor if not repaired. In this paper, the influence of both Sp diastereomers 4R and 4S as well as their anti and syn conformers on charge transfer through the double helix was taken into theoretical consideration. In addition, the electronic properties of four modelled double-stranded oligonucleotides (ds-oligos) were also discussed, i.e., d[A1Sp2A3oxoG4A5] * [T5C4T3C2T1]. Throughout the study, the M06-2X/6-31++G** level theory was used. Solvent-solute non-equilibrated and equilibrated interactions were also considered. The subsequent results elucidated that the 7,8-dihydro-8-oxo-guanosine:cytidine (OXOGC) base pair is the settled point of a migrated radical cation in each of the discussed cases, due to its low adiabatic ionization potential, i.e., ~5.55 [eV]. The opposite was noted for excess electron transfer through ds-oligos containing anti (R)-Sp or anti (S)-Sp. The radical anion was found on the OXOGC moiety, whereas in the presence of syn (S)-Sp or syn (R)-Sp, an excess electron was found on the distal A1T5 or A5T1 base pair, respectively. Furthermore, a spatial geometry analysis of the discussed ds-oligos revealed that the presence of syn (R)-Sp in the ds-oligo caused only a slight deformation to the double helix, while syn (S)-Sp formed an almost ideal base pair with a complementary dC. The above results are in strong agreement with the final charge transfer rate constant, as calculated according to Marcus' theory. In conclusion, DNA damage such as spirodi(iminohydantoin), especially when becoming part of clustered DNA damage, can affect the effectiveness of other lesion recognition and repair processes. This can lead to the acceleration of undesired and deleterious processes such as carcinogenesis or aging. However, in terms of anticancer radio-/chemo- or combined therapy, the slowing down of the repair machinery can result in increased effectiveness. With this in mind, the influence of clustered damage on charge transfer and its subsequent effect on single-damage recognition by glycosylases justifies future investigation.

Size- and Stereochemistry-Dependent Transcriptional Bypass of DNA Alkyl Phosphotriester Adducts in Mammalian Cells.

Environmental, endogenous and therapeutic alkylating agents can react with internucleotide phosphate groups in DNA to yield alkyl phosphotriester (PTE) adducts. Alkyl-PTEs are induced at relatively high frequencies and are persistent in mammalian tissues; however, their biological consequences in mammalian cells have not been examined. Herein, we assessed how alkyl-PTEs with different alkyl group sizes and stereochemical configurations (S P and R P diastereomers of Me and nPr) affect the efficiency and fidelity of transcription in mammalian cells. We found that, while the R P diastereomer of Me- and nPr-PTEs constituted moderate and strong blockages to transcription, respectively, the S P diastereomer of the two lesions did not appreciably perturb transcription efficiency. In addition, none of the four alkyl-PTEs induced mutant transcripts. Furthermore, polymerase η assumed an important role in promoting transcription across the S P-Me-PTE, but not any of other three lesions. Loss of other translesion synthesis (TLS) polymerases tested, including Pol κ, Pol ι, Pol ξ and REV1, did not alter the transcription bypass efficiency or mutation frequency for any of the alkyl-PTE lesions. Together, our study provided important new knowledge about the impact of alkyl-PTE lesions on transcription and expanded the substrate pool of Pol η in transcriptional bypass.

Chirality matters: stereo-defined phosphorothioate linkages at the termini of small interfering RNAs improve pharmacology in vivo.

A critical challenge for the successful development of RNA interference-based therapeutics therapeutics has been the enhancement of their in vivo metabolic stability. In therapeutically relevant, fully chemically modified small interfering RNAs (siRNAs), modification of the two terminal phosphodiester linkages in each strand of the siRNA duplex with phosphorothioate (PS) is generally sufficient to protect against exonuclease degradation in vivo. Since PS linkages are chiral, we systematically studied the properties of siRNAs containing single chiral PS linkages at each strand terminus. We report an efficient and simple method to introduce chiral PS linkages and demonstrate that Rp diastereomers at the 5′ end and Sp diastereomers at the 3′ end of the antisense siRNA strand improved pharmacokinetic and pharmacodynamic properties in a mouse model. In silico modeling studies provide mechanistic insights into how the Rp isomer at the 5′ end and Sp isomer at the 3′ end of the antisense siRNA enhance Argonaute 2 (Ago2) loading and metabolic stability of siRNAs in a concerted manner.

Probing the Catalytic Mechanism and Inhibition of SAMHD1 Using the Differential Properties of Rp- and Sp-dNTPαS Diastereomers.

SAMHD1 is a fundamental regulator of cellular dNTPs that catalyzes their hydrolysis into 2′-deoxynucleoside and triphosphate, restricting the replication of viruses, including HIV-1, in CD4+ myeloid lineage and resting T-cells. SAMHD1 mutations are associated with the autoimmune disease Aicardi-Goutières syndrome (AGS) and certain cancers. More recently, SAMHD1 has been linked to anticancer drug resistance and the suppression of the interferon response to cytosolic nucleic acids after DNA damage. Here, we probe dNTP hydrolysis and inhibition of SAMHD1 using the Rp and Sp diastereomers of dNTPαS nucleotides. Our biochemical and enzymological data show that the α-phosphorothioate substitution in Sp-dNTPαS but not Rp-dNTPαS diastereomers prevents Mg2+ ion coordination at both the allosteric and catalytic sites, rendering SAMHD1 unable to form stable, catalytically active homotetramers or hydrolyze substrate dNTPs at the catalytic site. Furthermore, we find that Sp-dNTPαS diastereomers competitively inhibit dNTP hydrolysis, while Rp-dNTPαS nucleotides stabilize tetramerization and are hydrolyzed with similar kinetic parameters to cognate dNTPs. For the first time, we present a cocrystal structure of SAMHD1 with a substrate, Rp-dGTPαS, in which an Fe–Mg-bridging water species is poised for nucleophilic attack on the Pα. We conclude that it is the incompatibility of Mg2+, a hard Lewis acid, and the α-phosphorothioate thiol, a soft Lewis base, that prevents the Sp-dNTPαS nucleotides coordinating in a catalytically productive conformation. On the basis of these data, we present a model for SAMHD1 stereospecific hydrolysis of Rp-dNTPαS nucleotides and for a mode of competitive inhibition by Sp-dNTPαS nucleotides that competes with formation of the enzyme–substrate complex.

Investigation of factors influencing the separation of diastereomers of phosphorothioated oligonucleotides

This study presents a systematic investigation of factors influencing the chromatographic separation of diastereomers of phosphorothioated pentameric oligonucleotides as model solutes. Separation was carried out under ion-pairing conditions using an XBridge C18 column. For oligonucleotides with a single sulfur substitution, the diastereomer selectivity was found to increase with decreasing carbon chain length of the tertiary alkylamine used as an ion-pair reagent. Using an ion-pair reagent with high selectivity for diastereomers, triethylammonium, it was found the selectivity increased with decreased ion-pair concentration and shallower gradient slope. Selectivity was also demonstrated to be dependent on the position of the modified linkage. Substitutions at the center of the pentamer resulted in higher diastereomer selectivity compared to substitutions at either end. For mono-substituted oligonucleotides, the retention order and stereo configuration were consistently found to be correlated, with Rp followed by Sp, regardless of which linkage was modified. The type of nucleobase greatly affects the observed selectivity. A pentamer of cytosine has about twice the diastereomer selectivity of that of thymine. When investigating the retention of various oligonucleotides eluted using tributylammonium as the ion-pairing reagent, no diastereomer selectivity could be observed. However, retention was found to be dependent on both the degree and position of sulfur substitution as well as on the nucleobase. When analyzing fractions collected in the front and tail of overloaded injections, a significant difference was found in the ratio between Rp and Sp diastereomers, indicating that the peak broadening observed when using tributylammonium could be explained by partial diastereomer separation.

Diastereomers of a mono-substituted phosphoryl guanidine trideoxyribonucleotide: Isolation and properties

Recently, a new type of nucleic acid analogues with modified phosphate group, namely, phosphoryl guanidine oligonucleotides, has been described. In the present work, we assess the difference between diastereomers of a mono-substituted phosphoryl guanidine oligonucleotide and analyze their resistance to nuclease digestion. Individual diastereomers (‘fast’ and ‘slow’) of a trideoxynucleotide d (TpCp*A) were isolated by reverse-phase HPLC. Snake venom phosphodiesterase digestion showed that the native trideoxynucleotide was fully degraded after 30 min, whereas both ‘fast’ and ‘slow’ diastereomers of d (TpCp*A) were not completely digested even after 7 days. UV and CD spectra revealed similarities in the structure of the diastereomers. Structural analysis by 1D and 2D NMR spectroscopy also uncovered significant similarity in the properties of Rp and Sp diastereomers. Structural analysis of nuclear Overhauser effect spectroscopy (NOESY) data and restrained molecular dynamics methods showed very flexible single-stranded oligonucleotide structures. Detailed computational analysis of restraint penalty energies via restrained molecular dynamics simulations with the 2D NMR interproton distance data allowed us to conclude that most likely, the ‘fast’ isomer is the Sp diastereomer, and the ‘slow’ isomer is the Rp diastereomer.

Reverse Transcription Past Products of Guanine Oxidation in RNA Leads to Insertion of A and C opposite 8-Oxo-7,8-dihydroguanine and A and G opposite 5-Guanidinohydantoin and Spiroiminodihydantoin Diastereomers.

Reactive oxygen species, both endogenous and exogenous, can damage nucleobases of RNA and DNA. Among the nucleobases, guanine has the lowest redox potential, making it a major target of oxidation. Although RNA is more prone to oxidation than DNA is, oxidation of guanine in RNA has been studied to a significantly lesser extent. One of the reasons for this is that many tools that were previously developed to study oxidation of DNA cannot be used on RNA. In the study presented here, the lack of a method for seeking sites of modification in RNA where oxidation occurs is addressed. For this purpose, reverse transcription of RNA containing major products of guanine oxidation was used. Extension of a DNA primer annealed to an RNA template containing 8-oxo-7,8-dihydroguanine (OG), 5-guanidinohydantoin (Gh), or the R and S diastereomers of spiroiminodihydantoin (Sp) was studied under standing start conditions. SuperScript III reverse transcriptase is capable of bypassing these lesions in RNA inserting predominantly A opposite OG, predominantly G opposite Gh, and almost an equal mixture of A and G opposite the Sp diastereomers. These data should allow RNA sequencing of guanine oxidation products by following characteristic mutation signatures formed by the reverse transcriptase during primer elongation past G oxidation sites in the template RNA strand.

New Lanthanide Tag for the Generation of Pseudocontact Shifts in DNA by Site-Specific Ligation to a Phosphorothioate Group.

Pseudocontact shifts (PCS) generated by paramagnetic lanthanides provide a rich source of long-range structural restraints that can readily be measured by nuclear magnetic resonance (NMR) spectroscopy. Many different lanthanide-binding tags have been designed for site-specific tagging of proteins, but established routes for tagging DNA with a single metal ion rely on difficult chemical synthesis. Here we present a simple and practical strategy for site-specific tagging of inexpensive phosphorothioate (PT) oligonucleotides. Commercially available PT oligonucleotides are diastereomers with S and R stereoconfiguration at the backbone PT site. The respective SP and RP diastereomers can readily be separated by HPLC. A new alkylating lanthanide-binding tag, C10, was synthesized that delivered quantitative tagging yields with both diastereomers. PCSs were observed following ligation with the complementary DNA strand to form double-stranded DNA duplexes. The PCSs were larger for the SP than the RP oligonucleotide and good correlation between back-calculated and experimental PCSs was observed. The C10 tag can also be attached to cysteine residues in proteins, where it generates a stable thioether bond. Ligated to the A28C mutant of ubiquitin, the tag produced excellent fits of magnetic susceptibility anisotropy (Δχ) tensors, with larger tensors than for the tagged PT oligonucleotides, indicating that the tag is not completely immobilized after ligation with a PT group.

Interrogation of Base Pairing of the Spiroiminodihydantoin Diastereomers Using the α-Hemolysin Latch.

Spiroiminodihydantoin (Sp) is a hyperoxidized form of guanine (G) resulting from oxidation by reactive oxygen species. The lesion is highly mutagenic, and the stereocenter renders the two isomers with distinct behaviors in chemical, spectroscopic, enzymatic, and computational studies. In this work, the α-hemolysin (αHL) latch sensing zone was employed to investigate the base pairing properties of the Sp diastereomers embedded in a double-stranded DNA. Duplexes containing (S)-Sp consistently gave deeper current blockage, and a baseline resolution of ∼0.8 pA was achieved between (S)-Sp:G and (R)-Sp:G base pairs. Ion fluxes were generally more hindered when Sp was placed opposite pyrimidines. Analysis of the current noise of blockade events further provided dynamics information about the Sp-containing base pairs. In general, base pairs comprised of (S)-Sp generated current fluctuations larger than those of their (R)-Sp counterparts, suggesting enhanced base pairing dynamics. The current noise was also substantially affected by the identity of the base opposite Sp, increasing in the following order: A < G < T < C. This report provides information about the dynamic structure of Sp in the DNA duplex and therefore has implications for the enzymatic repair of the Sp diastereomers.

Stereospecific Effects of Oxygen-to-Sulfur Substitution in DNA Phosphate on Ion Pair Dynamics and Protein-DNA Affinity.

Oxygen-to-sulfur substitutions in DNA phosphate often enhance affinity for DNA-binding proteins. Our previous studies have suggested that this effect of sulfur substitution of both OP1 and OP2 atoms is due to an entropic gain associated with enhanced ion pair dynamics. In this work, we studied stereospecific effects of single sulfur substitution of either the OP1 or OP2 atom in DNA phosphate at the Lys57 interaction site of the Antennapedia homeodomain-DNA complex. Using crystallography, we obtained structural information on the RP and SP diastereomers of the phosphoromonothioate and their interaction with Lys57. Using fluorescence-based assays, we found significant affinity enhancement upon sulfur substitution of the OP2 atom. Using NMR spectroscopy, we found significant mobilization of the Lys57 side-chain NH3 (+) group upon sulfur substitution of the OP2 atom. These data provide further mechanistic insights into the affinity enhancement by oxygen-to-sulfur substitution in DNA phosphate.

In Vitro Selection of a DNAzyme Cooperatively Binding Two Lanthanide Ions for RNA Cleavage.

Trivalent lanthanide ions (Ln(3+)) were recently employed to select RNA-cleaving DNAzymes, and three new DNAzymes have been reported so far. In this work, dysprosium (Dy(3+)) was used with a library containing 50 random nucleotides. After six rounds of in vitro selection, a new DNAzyme named Dy10a was obtained and characterized. Dy10a has a bulged hairpin structure cleaving a RNA/DNA chimeric substrate. Dy10a is highly active in the presence of the five Ln(3+) ions in the middle of the lanthanide series (Sm(3+), Eu(3+), Gd(3+), Tb(3+), and Dy(3+)), while its activity descends on the two sides. The cleavage rate reaches 0.6 min(-1) at pH 6 with just 200 nM Sm(3+), which is the fastest among all known Ln(3+)-dependent enzymes. Dy10a binds two Ln(3+) ions cooperatively. When a phosphorothioate (PS) modification is introduced at the cleavage junction, the activity decreases by >2500-fold for both the Rp and Sp diastereomers, and thiophilic Cd(2+) cannot rescue the activity. The pH-rate profile has a slope of 0.37 between pH 4.2 and 5.2, and the slope was even lower at higher pH. On the basis of these data, a model of metal binding is proposed. Finally, a catalytic beacon sensor that can detect Ho(3+) down to 1.7 nM is constructed.

Effect of Base-Pairing Partner on the Thermodynamic Stability of the Diastereomeric Spiroiminodihydantoin Lesion.

Oxidation of guanine by reactive oxygen species and high valent metals produces damaging DNA base lesions like 8-oxo-7,8-dihydroguanine (8-oxoG). 8-oxoG can be further oxidized to form the spiroiminodihydantoin (Sp) lesion, which is even more mutagenic. DNA polymerases preferentially incorporate purines opposite the Sp lesion, and DNA glycosylases excise the Sp lesion from the duplex, although the rate of repair is different for the two Sp diastereomers. To further understand the biological processing of the Sp lesion, differential scanning calorimetry studies were performed on a series of 15-mer DNA duplexes. The thermal and thermodynamic stabilities of each of the Sp diastereomers paired to the four standard DNA bases were investigated. It was found that, regardless of the base-pairing partner, the Sp lesion was always highly destabilizing in terms of DNA melting temperature, enthalpic stability, and overall duplex free energy. We found no significant differences between the two Sp diastereomers, but changing the base-pairing partner of the Sp lesion produced slight differences in stability. Specifically, duplexes with Sp:C pairings were always the most destabilized, whereas pairing the Sp lesion with a purine base modestly increased stability. Overall, these results suggest that, although the stability of the Sp diastereomers cannot explain the differences in the rates of repair by DNA glycosylases, the most stable base-pairing partners do correspond with the nucleotide preference of DNA polymerases.

Synthesis, Hydrolysis, and Protonation-Promoted Intramolecular Reductive Breakdown of Potential NRTIs: Stavudine α-P-Borano-γ-P-N-L-tryptophanyltriphosphates.

Phosphorus-modified prodrugs of dideoxynucleoside triphosphates (ddNTPs) have shown promise as pronucleotide strategies for improving antiviral activity compared to their parent dideoxynucleosides. Borane modified NTPs offer a promising choice as nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs). However, the availability of α-P-borano-γ-P-substituted NTP analogs remains limited due to challenges with synthesis and purification. Here, we report the chemical synthesis and stability of a new potential class of NRTI prodrugs: stavudine (d4T) 5′-α-P-borano-γ-P-N-l-tryptophanyltriphosphates. One-pot synthesis of these compounds was achieved via a modified cyclic trimetaphosphate approach. Pure Rp and Sp diastereomers were obtained after HPLC separation. Based on LC-MS analysis, we report degradation pathways, half-lives (5–36 days) and mechanisms arising from structural differences to generate the corresponding borano tri- and di-phosphates, and H-phosphonate, via several parallel routes in buffer at physiologically relevant pH and temperature. Here, the major hydrolysis products, d4T α-P-boranotriphosphate Rp and Sp isomers, were isolated by HPLC and identified with spectral data. We first propose that one of the major degradation products, d4T H-phosphonate, is generated from the d4T pronucleotides via a protonation-promoted intramolecular reduction followed by a second step nucleophilic attack. This report could provide valuable information for pronucleotide-based drug design in terms of selective release of target nucleotides.

Convenient synthesis of nucleoside 5&amp;prime;-(&amp;alpha;-P-thio)triphosphates and phosphorothioate nucleic acids (DNA and RNA)

Modified deoxy- and ribo-nucleoside triphosphates are chemically synthesized in multiple steps due to the protection and deprotection of the nucleoside functionalities. To conveniently synthesize the S -modified triphosphates for enzymatically preparing phosphorothioate DNAs and RNAs (PS-DNA and PS-RNA) as potential therapeutics, herein we report a one-pot strategy to synthesize the deoxy- and ribo-nucleoside 5′-(α-P-thio)triphosphates (dNTPαS and NTPαS) without protecting any nucleoside functionalities. This facile synthesis is achieved by treating the nucleosides with a mild phosphitylating reagent, reacting selectively with the 5′-hydroxyl group of each unprotected nucleoside, followed by sulfurization and hydrolysis to afford the crude dNTPαS and NTPαS analogs (mixtures of Sp and Rp diastereomers). We also demonstrated that after just simple precipitation (without HPLC and ion-exchange purification), the quality of the synthesized dNTPαS and NTPαS analogs is excellent for direct DNA polymerization and RNA transcription, respectively. Since Klenow DNA polymerase and T7 RNA polymerase accept the Sp diastereomers of dNTPαS and NTPαS analogs, respectively, while the Rp diastereomers are neither substrates nor inhibitors, the diastereomerically-pure PS-DNAs and PS-RNAs can be conveniently synthesized enzymatically.

Interaction and Positioning of Nucleosome Core Particles on DNA Containing the Guanine-Derived Oxidative Lesion Spiroiminodihydantoin

Oxidative DNA damage has been implicated in cellular aging, neurological disorders, and carcinogenesis. Living in an oxidizing environment makes DNA damage unavoidable, and some estimates suggest that human DNA withstands as many as 10,000 oxidative events per cell per day. One of the most widely studied DNA lesions is 7,8-dihydro-8-oxo-2’-deoxyguanosine (8-oxoG). Interestingly, the 8-oxoG lesion has a lower reduction potential than guanine and the other DNA bases, allowing it to react further to produce additional base lesions including the spiroiminodihydantoin (Sp) lesion.The Sp lesion contains two rigid five-membered rings connected via a central chiral carbon. As a result, oxidation of 8-oxoG leads to the formation of a pair of Sp diastereomers, both of which may impact the stability and functionality of genomic DNA. Both Sp diastereomers are highly mutagenic, leading to transversions. Though no structures of DNA containing the Sp lesion have yet been determined, computational studies predict that the Sp lesion disrupts hydrogen bonding and base stacking and causes groove widening, therefore disrupting the stability of the DNA double helix. Thermodynamic studies have shown that the Sp lesion destabilizes the DNA double helix by 3.6-6.7 kcal/mol, depending on the nucleotide sequence.In our current work we seek to better understand the effects of the Sp adduct as it effects packaging and positioning of a DNA sequence on nucleosomes. The locations of the histone octamer on DNA molecules are strongly influenced by nucleotide sequence and DNA structure, and this positioning is critically linked to gene expression and gene silencing. Given that the Sp lesion is likely to significantly distort helix structure, we expect the lesion to alter DNA-histone contacts generating changes to the rotational and translational setting of positioned nucleosomes.

Sequence-Specific Stereomeric Environment in a DNA Duplex Revealed by a Nucleotide-Independent Nitroxide Probe

In site-directed spin labeling, structural and dynamic information on a parent macromolecule is obtained by monitoring a covalently linked nitroxide radical using electron paramagnetic resonance (EPR) spectroscopy. Our group have developed a method of attaching nitroxide species, such as 1-oxyl-4-bromo-2,2,5,5-tetramethylpyrroline (R5a), to a specific nucleotide position within a target DNA or RNA sequence. The method relies on site-specific introduction of a phosphorothiate during the solid phase chemical synthesis of nucleic acids, and at each given labeling site the nitroxide is attached to one of two phosphorothioate diastereomers (Rp or Sp) in an approximately 50/50 ratio. We have recently reported that variations in DNA structural and dynamic features at the level of an individual nucleotide can be detected using R5a attached to mixed phosphorothioate diastereomers, in which an observed EPR spectrum is presumably a sum of those obtained from either diastereomer (Popova et al., Biochemistry, 2009, 48, 8540-50). In this work, we report X-band EPR spectra of R5a attached to purified Rp and Sp diastereomers at different sites within a B-form DNA duplex. Results are compared to those obtained with mixed nitroxide diastereomers, and advantages and limitations are discussed regarding the necessity of diastereomer separation when probing DNA local environment. Our work is a further step forward in developing a SDSL methodology that may provide a mean for studying structure and dynamics in large DNA molecules.

Synthesis and characterization of mRNA cap analogs containing phosphorothioate substitutions that bind tightly to eIF4E and are resistant to the decapping pyrophosphatase DcpS

Analogs of the mRNA cap are widely employed to study processes involved in mRNA metabolism as well as being useful in biotechnology and medicinal applications. Here we describe synthesis of six dinucleotide cap analogs bearing a single phosphorothioate modification at either the alpha, beta, or gamma position of the 5',5'-triphosphate chain. Three of them were also modified with methyl groups at the 2'-O position of 7-methylguanosine to produce anti-reverse cap analogs (ARCAs). Due to the presence of stereogenic P centers in the phosphorothioate moieties, each analog was obtained as a mixture of two diastereomers, D1 and D2. The mixtures were resolved by RP HPLC, providing 12 different compounds. Fluorescence quenching experiments were employed to determine the association constant (K(AS)) for complexes of the new analogs with eIF4E. We found that phosphorothioate modifications generally stabilized the complex between eIF4E and the cap analog. The most strongly bound phosphorothioate analog (the D1 isomer of the beta-substituted analog m(7)Gpp(S)pG) was characterized by a K(AS) that was more than fourfold higher than that of its unmodified counterpart (m(7)GpppG). All analogs modified in the gamma position were resistant to hydrolysis by the scavenger decapping pyrophosphatase DcpS from both human and Caenorhabditis elegans sources. The absolute configurations of the diastereomers D1 and D2 of analogs modified at the alpha position (i.e., m(7)Gppp(S)G and m(2) (7,2'-O )Gppp(S)G) were established as S(P) and R(P) , respectively, using enzymatic digestion and correlation with the S(P) and R(P) diastereomers of guanosine 5'-O-(1-thiodiphosphate) (GDPalphaS). The analogs resistant to DcpS act as potent inhibitors of in vitro protein synthesis in rabbit reticulocyte lysates.