UV Thermal Denaturation Research Articles

Locked Nucleic Acid Oligonucleotides Facilitate RNA•LNA-RNA Triple-Helix Formation and Reduce MALAT1 Levels.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and multiple endocrine neoplasia-β (MENβ) are two long noncoding RNAs upregulated in multiple cancers, marking these RNAs as therapeutic targets. While traditional small-molecule and antisense-based approaches are effective, we report a locked nucleic acid (LNA)-based approach that targets the MALAT1 and MENβ triple helices, structures comprised of a U-rich internal stem-loop and an A-rich tract. Two LNA oligonucleotides resembling the A-rich tract (i.e., A9GCA4) were examined: an LNA (L15) and a phosphorothioate LNA (PS-L15). L15 binds tighter than PS-L15 to the MALAT1 and MENβ stem loops, although both L15 and PS-L15 enable RNA•LNA-RNA triple-helix formation. Based on UV thermal denaturation assays, both LNAs selectively stabilize the Hoogsteen interface by 5-13 °C more than the Watson-Crick interface. Furthermore, we show that L15 and PS-L15 displace the A-rich tract from the MALAT1 and MENβ stem loop and methyltransferase-like protein 16 (METTL16) from the METTL16-MALAT1 triple-helix complex. Human colorectal carcinoma (HCT116) cells transfected with LNAs have 2-fold less MALAT1 and MENβ. This LNA-based approach represents a potential therapeutic strategy for the dual targeting of MALAT1 and MENβ.

Global RNA modifications to the MALAT1 triple helix differentially affect thermostability and weaken binding to METTL16

Therapeutic mRNAs are generated using modified nucleotides, namely N1-methylpseudouridine (m1Ψ) triphosphate, so that the mRNA evades detection by the immune system. RNA modifications, even at a single-nucleotide position, perturb RNA structure, although it is not well understood how structure and function is impacted by globally modified RNAs. Therefore, we examined the metastasis-associated lung adenocarcinoma transcript 1 triple helix, a highly structured stability element that includes single-, double-, and triple-stranded RNA, globally modified with N6-methyladenosine (m6A), pseudouridine (Ψ), or m1Ψ. UV thermal denaturation assays showed that m6A destabilizes both the Hoogsteen and Watson-Crick faces of the RNA by ∼20 °C, Ψ stabilizes the Hoogsteen and Watson-Crick faces of the RNA by ∼12 °C, and m1Ψ has minimal effect on the stability of the Hoogsteen face of the RNA but increases the stability of the Watson-Crick face by ∼9 °C. Native gel-shift assays revealed that binding of the methyltransferase-like protein 16 to the metastasis-associated lung adenocarcinoma transcript 1 triple helix was weakened by at least 8-, 99-, and 23-fold, respectively, when RNA is globally modified with m6A, Ψ, or m1Ψ. These results demonstrate that a more thermostable RNA structure does not lead to tighter RNA-protein interactions, thereby highlighting the regulatory power of RNA modifications by multiple means.

Mitochondriotropic agents conjugated with NSAIDs through metal ions against breast cancer cells

Two copper(I) polymorphs of formula [Cu(SALH)(TPP)3] (1a and 1b) were prepared by the conjugation of the Non-Steroidal Anti-Inflammatory Drug (NSAID) salicylic acid (SALH2) with the mitochondriotropic agent triphenylphosphine (TPP) via metal ion. For comparison, the isomorph [Ag(SALH)(TPP)3] (2) was prepared. The conjugates 1a, 1b and 2 were characterized by melting point (m.p.), Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy, Ultraviolet-Visible (UV–Vis) spectroscopy and nuclear magnetic resonance (1H NMR). The crystal structures of 1a, 1b and 2 were confirmed by X-ray diffraction crystallography (XRD). The ex vivo binding affinity of 1–2 towards CT (calf thymus)-DNA was studied by UV, fluorescence, viscosity and DNA Thermal Denaturation studies. Their inhibitory activity against lipoxygenase (LOX) (an enzyme which is mainly located in the mitochondrion) was determined. The in vitro activity of 1–2 was evaluated against human breast cancer cell lines MCF-7 (hormone depended (HD)) and MDA-MB 281 (hormone independent (HI)) cells. Compounds 1–2 inhibit stronger than cisplatin the cancerous cells. The molecular mechanism of action of 1–2 was suspected by the MCF-7 cells morphology and confirmed by DNA fragmentation, Acridine Orange/Ethidium Bromide (AO/EB) Staining and mitochondrial membrane permeabilization tests.

Identification of G4 motifs of various stem cell markers and their biophysical and biochemical characterization

Regulatory regions in the human genome, enriched in guanine-rich DNA sequences have a remarkable enrichment of G-rich sequences having a tendency to fold into G-quadruplex structures. To identify the G-quadruplex forming motifs in regulatory regions of stem cell markers, gene sequences of various stem cell markers were downloaded and analyzed to see the abundance of G-rich sequences. We observed the enrichment of G-rich sequences in stem cell markers (CD13, CD19, CD24 and CD38) which could possibly play a critical role in its regulation. We used Circular Dichroism (CD), UV-Thermal denaturation (UV-T m) and polyacrylamide gel electrophoresis (PAGE) to demonstrate the formation of a G-quadruplex by G-rich sequences present in these stem cell markers. We observed that these G-rich sequences containing minimum consecutive G3 stretch separated by loop length ranging from one to three bases long adopt G-quadruplexes with different molecularity involving two-strands, three-strand and four-strand with parallel and antiparallel conformation. Interestingly, we proposed the formation of three-stranded G-quadruplex by CD13 in 100 mM Na+, CD19 in 100 mM K+, 100 mM K+ with 40 wt% PEG 200, and CD38 in 100 mM K+ + 40 wt% PEG 200. The formation of such diverse G-quadruplex structures in the regulatory regions leaves the fair possibility of recognition by regulatory factors to modulate the gene expression. First time, this study may give insight into the structural polymorphism of G4 forming motifs in different stem cell markers to design the best suitable ligand and to target them for therapeutic development. Communicated by Ramaswamy H. Sarma

Protein-RNA interactions of FMRP and CCND1: Implications of regulation in neurogenesis.

While originally discovered in the 1960s, adult neurogenesis, the production of mature neurons from neuronal stem cells in adults, has become largely accepted by the field only in the 1990s. Thus, the detailed mechanisms of its regulation remain elusive, requiring further characterization of the protein players involved in this process. Cyclin-D1, a regulatory subunit of cyclin-dependent kinases involved in monitoring entry into the cell cycle, has been identified as a key factor in adult neurogenesis and is encoded by the CCND1 gene. Another identified component of this process is the Fragile X messenger ribonucleoprotein (FMRP), an RNA-binding protein associated with translational regulation. Interestingly, FMRP has been shown to regulate cyclin-D1 expression, emphasizing a potential interplay between FMRP function and cyclin-D1 expression in neurons. FMRP has shown high affinity binding for guanine quadruplex (GQ) forming RNAs. We identified that CCND1 mRNA has a G-rich region in its 3’-untranslated region that has the potential to form GQ structures, and thus possibly interacting with FMRP through the recognition of these structures. To elucidate the presence of a GQ in the CCND1 mRNA, we used biophysical methods, such as 1H NMR, UV thermal denaturation, CD spectroscopy, and native polyacrylamide gel electrophoresis (PAGE). This study effectively characterizes a GQ in the 3’UTR of the CCND1 mRNA and its interactions with FMRP, providing insight on additional regulatory mechanisms of adult neurogenesis and guiding development of novel therapeutic methods.

Impact of the zinc complexation of polytopic polyaza ligands on the interaction with double and single stranded DNA/RNA and antimicrobial activity.

Metal complexes have gained a huge interest in the biomedical research in the last decade because of the access to unexplored chemical space with regards to organic molecules and to present additional functionalities to act simultaneously as diagnostic and therapeutic agents. Herein, we evaluated the interaction of two polytopic polyaza ligands and their zinc complexes with DNA and RNA by UV thermal denaturation, fluorescence and circular dichroism spectroscopic assays. The zinc coordination was investigated by X-ray diffraction and afforded the structure of the binuclear zinc complex of PYPOD. Thermal denaturation of DNA and RNA and fluorimetry analysis revealed preferential binding of the zinc-PHENPOD complexes towards GC-containing DNA in contrast to the free ligands. On the other hand, PYPOD metal complexes, compared to the free ligand, stabilized AT-based DNA (B-form) better than AU-RNA (A-form). With regards to single stranded RNA, the binuclear complex of PHENPOD and the free ligand can efficiently identify polyadenylic acid (poly A) among other RNA sequences by circular dichroism spectroscopy. The antimicrobial activity in S. aureus and E. coli bacteria showed the highest activity for the free ligands and their trinuclear zinc complexes. This work can provide valuable insights into the impact of the nuclearity of polytopic polyaza ligands in the binding to DNA/RNA and the antimicrobial effect.

Substituent effects on indole universal bases in DNA

A series of eleven 5-substituted-indole nucleoside residues were synthesized and incorporated into 15-mer oligodeoxynucleotide duplexes with each of the four natural bases (A, G, C, T) paired with the non-natural residues. The universal base properties of these residues were evaluated by comparing the stabilities of these complexes determined by UV thermal denaturation experiments. All of the substituted indoles were more stable than an unsubstituted indole residue, and measures of electronic influences of substituents on the indole pi system correlate with stability. Several residues were more promiscuous than the previously studied 5-nitroindole residue, but the relationship between substituents and selectivity is not clear.

A novel cataract-causing mutation Ile82Met of γA crystallin trends to aggregate with unfolding intermediate

Cataract is the most common pathogenic ophthalmic disease leading to blindness in children worldwide. Genetic disorder is the leading cause of congenital cataract, among which crystallin mutations have a high incidence. There are few reports on γA-crystallin, one critical member of crystallin superfamilies. In this study, we identified a novel pathogenic mutation (Ile82Met) in γA-crystallin from a three-generation Chinese family with cataract, and investigated the potential molecular mechanism in detail. To elucidate the pathogenic mechanism of I82M mutant, spectroscopic and solubility experiments were performed to determine the difference between the purified γA-crystallin wild type (WT) and I82M mutant under both physiological conditions and environmental stresses (UV irradiation, thermal denaturation or chemical denaturation). The I82M mutant did not affect the secondary/tertiary structure of monomeric γA-crystallin under physiological status, but decreased protein stability and increased aggregatory potency under the stressful treatment. Surprisingly, the chemical denaturation caused I82M to switch from the two-state unfolding of γA-crystallin to three-state unfolding involving an unfolding intermediate. This study expands the genetic variation map of cataract, and provides novel insights into the pathomechanism, in particular, filling in a gap in the understanding of γA-crystallin mutants causing cataract.

Novel Pyridazin-3(2H)-one-Based Guanidine Derivatives as Potential DNA Minor Groove Binders with Anticancer Activity.

Novel aryl guanidinium analogues containing the pyridazin-3(2H)-one core were proposed as minor groove binders (MGBs) with the support of molecular docking studies. The target dicationic or monocationic compounds, which show the guanidium group at different positions of the pyridazinone moiety, were synthesized using the corresponding silyl-protected pyridazinones as key intermediates. Pyridazinone scaffolds were converted into the adequate bromoalkyl derivatives, which by reaction with N,N’-di-Boc-protected guanidine followed by acid hydrolysis provided the hydrochloride salts 1–14 in good yields. The ability of new pyridazin-3(2H)-one-based guanidines as DNA binders was studied by means of DNA UV-thermal denaturation experiments. Their antiproliferative activity was also explored in three cancer cell lines (NCI-H460, A2780, and MCF-7). Compounds 1–4 with a bis-guanidinium structure display a weak DNA binding affinity and exhibit a reasonable cellular viability inhibition percentage in the three cancer cell lines studied.

Small Molecule Screening Discovers Compounds that Reduce FMRpolyG Protein Aggregates and Splicing Defect Toxicity in Fragile X-Associated Tremor/Ataxia Syndrome.

Expansion of CGG trinucleotide repeats in 5' untranslated region of the FMR1 gene is the causative mutation of neurological diseases such as fragile X syndrome (FXS), fragile X-associated tremor/ataxia syndrome (FXTAS), and ovarian disorder such as fragile X-associated primary ovarian insufficiency (FXPOI). CGG repeats containing FMR1 transcripts form the toxic ribonuclear aggregates, abrupt pre-mRNA splicing, and cause repeat-associated non-AUG translation, leading to the disease symptoms. Here, we utilized a small molecule library of ~ 250,000 members obtained from the National Cancer Institute (NCI) and implemented a shape-based screening approach to identify the candidate small molecules that mitigate toxic CGG RNA-mediated pathogenesis. The compounds obtained from screening were further assessed for their affinity and selectivity towards toxic CGG repeat RNA by employing fluorescence-binding experiment and isothermal calorimetry titration assay. Three candidate molecules B1, B4, and B11 showed high affinity and selectivity for expanded CGG repeats RNA. Further, NMR spectroscopy, gel mobility shift assay, CD spectroscopy, UV-thermal denaturation assay, and molecular docking affirmed their high affinity and selectivity for toxic CGG RNAs. Next, these lead compounds selectively improved the pre-mRNA alternative splicing defects with no perturbation in global splicing efficacy and simultaneouslyreduced the FMR1polyG protein aggregate formation without affecting the downstream expression of the gene. Taken together these findings, we addressed compound B1, B4, and B11 as potential lead molecules for developing promising therapeutics against FXTAS. Herein, this study, we have utilized shape similarity approach to screen the NCI library and found out the potential candidate which improves the pre-mRNA splicing defects and reduces FMR1polyG aggregations.

Structural switching/polymorphism by sequential base substitution at quasi-palindromic SNP site (G → A) in LCR of human β-globin gene cluster

The human β-globin gene Locus Control Region (LCR), a dominant regulator of globin gene expression contains five tissue-specific DNase I-hypersensitive sites (HSs). A single nucleotide polymorphism (SNP) (A → G) present in HS4 region of locus control region (LCR), have shown a notable association between the G allele and the occurrence of β-thalassemia. This SNP site exhibiting a hairpin − duplex equilibrium manifested in A → B like DNA transition has previously been reported from this laboratory. Since, DNA is a dynamic and adaptable molecule, so any change of a single base within a primary DNA sequence can produce major biological consequences commonly manifested in genetic disorders such as sickle cell anemia and β-thalassemia. Herein, the differential behavior of sequential single base substitutions G → A on the quasi-palindromic sequence (d-TGGGGGCCCCA; HPG11) has been explored. A combination of native gel electrophoresis, circular dichroism (CD), and UV-thermal denaturation (Tm) techniques have been used to investigate the structural polymorphism associated with various variants of HPG11 i.e. HPG11A2 to HPG11A5. The CD spectra confirmed that all the HPG11 variants exhibit a hairpin − duplex equilibrium. Oligomer concentration dependence on CD spectra has been correlated with A → B DNA conformational transition. However, as revealed in gel electrophoresis, HPG11A2 → A5 exhibit the formation of a tetramolecular structure (four-way junction) at higher oligomer concentration. UV-melting studies also supported the melting of hairpin, duplex and four-way junction structure. This polymorphism pattern may possibly be significant for DNA-protein recognition, in the process of regulation of LCR in the β-globin gene.

Influence of C5‐Substituents on Repair of O4‐Methyl Adducts of Pyrimidines by O6‐Alkylguanine DNA Alkyltransferases

AbstractThe DNA repair protein O6‐alkylguanine‐DNA alkyltransferase (AGT), found in numerous organisms, can remove methyl groups from the O6‐ and O4‐atoms of 2′‐deoxyguanosine and thymidine in DNA. AGT variants demonstrate different repair efficiencies towards these lesions. To understand the influence of C5 nucleobase substituents on O4‐methyl removal by AGTs, DNA duplexes containing 5‐chloro‐, 5‐bromo‐ 5‐iodo‐ and 5‐trifluoromethyl‐O4‐methyl‐2′‐deoxyuridine were studied. UV thermal denaturation revealed a stability reduction of 11 °C for the O4‐methyl halogen series and 5‐trifluoromethyl analog relative to their controls. For the 5‐chloro analog efficient repair was observed by human and E.coli AGTs. For the larger halogens (5‐bromo and 5‐iodo) and 5‐trifluoromethyl analog, human AGT showed moderate repair of the O4‐methyl adduct. E.coli OGT and Ada‐C readily repaired most adducts with reduced efficiency for the larger groups, except C5‐iodo. These results suggest electronic contributions and favourable interactions of the C5‐halogens within the AGT active site contribute to efficient dealkylation.

Terpene Chain Length Affects the Base Pairing Discrimination of S-geranyl-2-thiouridine in RNA Duplex.

SummaryGeranylation is a hydrophobic modification discovered in several bacteria tRNAs with the function of promoting codon bias during translation. However, why nature selects this C10-geranyl group remains a question. We conduct synthesis, UV-thermal denaturation, and molecular simulation studies in RNA duplexes and reveal possible reasons behind this natural selection. Among methyl-(C1), dimethylallyl-(C5), geranyl-(C10), and farnesyl-(C15) modified 2-thiouridines, only geranyl-group promotes U:G over U:A pair. Molecular simulation shows all the modified terpene groups point to the minor groove of RNA duplexes. The discrimination between U:G and U:A pairs derives from the difference in hydrogen bonding and interactions of the chain with the hydrophobic area in the minor groove. Geranyl group has perfect length to discriminate U:G and U:A pairs, whereas the others are either too long or too short to achieve the same behavior. This work indicates that geranyl group cannot be replaced by other terpene groups in promoting codon-specificity.

The Effect of Divalent Metal Ions on the Temperature Stability of Poly(I:C) Duplex

The effect of divalent ions on the thermal stability of the double-stranded polynucleotide polyinosinic:polycytidylic acid (poly(I:C)) in a buffer solution (pH 7) was studied by UV absorption spectroscopy and thermal denaturation by measuring the absorbance at 260 nm. The concentration dependence of the polynucleotide melting temperature (Tm) in the presence of metal ions (2.5 × 10−6–10−2 M) was obtained. It was found that the duplex thermal stability grew gradually upon an increase of Ca2+ and Mn2+ concentration. The thermal stability of the duplex in the presence of Cd2+, Ni2+, and Zn2+ rises up to an ion concentration of about 10−4 M and further falls. In contrast to these ions, the addition of Cu2+ to the solution leads to a sharp decline in Тm of the duplex at a concentration above 10−5 M. The observed difference in the influence of metal ions on the poly(I:C) thermal stability is explained by the different affinity of these ions to both the phosphate groups and the nitrogenous bases of the polynucleotides. Based on the analysis of the Tm concentration dependence, the dominant binding of each ion to the specific polynucleotide components was proposed.

Formation of a DNA triple helical structure at BOLF1 gene of human herpesvirus 4 (HH4) genome

Eukaryotic genomes contain a large number of pyrimidine-purine rich regions and such regions can assume varied DNA conformations, including triple-stranded structures. These structures have fascinated scientists because of their considerable therapeutic applications. These structures have also profound implications in the field of nanotechnology as they can be used to develop DNA-based nanostructures and materials. Therefore, for any application, it is important to understand the formation of triplex structures, both in quantitative and qualitative terms. A combination of gel electrophoresis, UV-thermal denaturation and circular dichroism (CD) spectroscopy was used to investigate the formation of inter- as well as intramolecular triplex, in pyrimidine motif at BOLF1 gene of human herpesvirus 4 (HH4) genome. This gene codes for inner tegument protein, which plays crucial roles in viral replication. The said oligopurine•oligopyrimidine duplex was targeted via a designed triple helix forming oligopyrimidine nucleotide (TFO) in intermolecular as well as intramolecular fashion. Our studies revealed that intramolecular triplex formation takes place at acidic as well as at neutral pH; whereas low pH is required for its intermolecular version. This comparative study between inter- and intramolecular triplex allowed us to demonstrate that intramolecular structure is more stable to its intermolecular counterpart. Numerous models for mono-, bi- and trimolecular structures adopted by these DNA sequences have been suggested. This report adds to our existing knowledge about DNA triple helical structures.

Cationic porphyrins as destabilizer of a G-quadruplex located at the promoter of human MYH7 β gene

G-quadruplex (GQ) architecture is adopted by guanine rich sequences, present throughout the eukaryotic genome including promoter locations and telomeric ends. The in vivo presence indicates their involvement and role in various biological processes. Various small ligands have been developed to interact and stabilize/destabilize G-quadruplex structures. Cationic porphyrins are among the most studied ligands, reported to bind and stabilize G-quadruplexes. Herein, we report the recognition and destabilization of a parallel G-quadruplex by porphyrins (TMPyP3 and TMPyP4). This G-quadruplex forming 23-nt G-rich sequence is in the promoter region of Human Myosin Heavy Chain β gene (MYH7β). Presence of various putative regulatory sequence elements (TATA Box, CCAAT, SP-1) located in the vicinity of this quadruplex motif, highlight its regulatory implications. Biophysical methods as Circular Dichroism Spectroscopy, UV-Absorption Spectroscopy, UV-Thermal Denaturation and Fluorescence Spectroscopy (steady as well as Time Resolved) have been used for studying the interaction and binding parameters. It is proposed that porphyrins have a destabilizing effect on the G-quadruplexes with parallel topology and a stronger binding specifically via intercalation mode is needed to cause destabilization. The study deals with better understanding and insights of DNA-Drug interactions in biological systems.Communicated by Ramaswamy H. Sarma

Elucidation of binding interactions and mechanism of Fludarabine with dsDNA via multispectroscopic and molecular docking studies

Fludarabine is a purine derivative, anti-neoplastic drug and is still being used in the treatments of chronic lymphocytic leukemia, small lymphocytic lymphoma, acute myeloid leukemia, Non-Hodgkin’s lymphoma. It achieves its function by interacting with DNA. Therefore, the binding interactions of such drugs with deoxyribonucleic acid (DNA) is an important subject for pharmaceutical and biochemical studies aiming at designing better DNA binding drugs. Although DNA binding mode of some of the anti-neoplastic drugs has been studied, DNA interaction of Fludarabine has not been explored yet. For this reason, this work has been dedicated to deciphering the experimental and theoretical investigation of Fludarabine binding mechanism via multispectroscopic techniques including UV absorption spectroscopy, thermal denaturation, fluorescence and FTIR spectroscopy, electrochemical and viscosity measurement methods as well as with molecular docking studies under physiological conditions. We observed in the lowest energy docking poses that Fludarabine binds to DNA via major groove binding mode. The nonplanar and extended structure and hydrogen bonding interactions of Fludarabine with the Adenine-Thymine base-pair played a very decisive role in the binding mode as supported by the experimental results.

Deciphering the Binding Mechanism of Noscapine with Lysozyme: Biophysical and Chemoinformatic Approaches.

Lysozyme is a well-characterized protein in terms of its structure, dynamics, and functions. It has thus emerged as a potential target to understand protein–drug interactions. The aim of our study is to gain a biophysical outlook on the interaction of lysozyme (Lyz), a well-known model protein, with Noscapine, a potent tubulin-binding anticancer drug. Noscapine (Nos) is effective against a wide range of cancer and shows low toxicity and few side effects. We report the underlying mechanism of complex formation between Nos and Lyz using spectroscopic and advanced computational avenues. The spectroscopic techniques, that is, absorption and steady-state and time-resolved fluorescence, proved that Lyz–Nos forms a complex, and the quenching mechanism was of the static type. The binding constant was in the order of 103 indicative of moderate binding, while the stoichiometry of the protein–drug complex was 1:1 at 298 K. The secondary structural analysis using CD and UV thermal denaturation further confirmed the conformational changes in the protein upon binding with Nos. Molecular dynamics simulation studies confirmed the stable binding with minimum deviations in RMSD. The above conclusions are significant to the development of the pharmacokinetics and pharmacodynamic properties of Nos, and its successful interaction with a versatile protein like Lyz will help in overcoming its previous limitations.

Structure-activity relationships of new Organotin(IV) anticancer agents and their cytotoxicity profile on HL-60, MCF-7 and HeLa human cancer cell lines

There is a growing interest in the cancer cell growth inhibitory effects of organotin (IV) compounds and, accordingly, a new series of dimethyl-, di-(n-butyl)-, diphenyl- and chloro-phenyl tin(IV) complexes with a Schiff base core were prepared. Their binding to DNA was assessed by UV thermal denaturation showing no interaction and by UV–vis titration exhibiting moderate interaction by intercalation. Complexes having n-butyl substituents were more potent and cytotoxic against human leukemia, breast and cervical cancer cell lines than other organotin(IV) complexes tested. Unfortunately, some of these compounds showed similar cytotoxicity in a non-cancerous cell line. We may conclude that cytotoxic activity was dependent on the nature (lipophilicity and size, according to the structure-activity relationship studies) and substitution pattern on the different structures. These results may aid in the rational design of metallodrugs, expanding the scope of organotin complexes in formulating new metal based drugs with dibutyl moieties.

Adduct Fluorescence as a Tool to Decipher Sequence Impact on Frameshift Mutations Mediated by a C-Linked C8-Biphenyl-Guanine Lesion.

Aromatic chemicals can undergo metabolic activation to afford electrophilic species that react at the C8-site of 2'-deoxyguanosine (dG) to generate bulky C8-dG adducts as a basis of initiating carcinogenesis. These DNA lesions have served as models to understand the mechanism of frameshift mutagenesis, especially within CG-dinucleotide repeat sequences, such as NarI (5'-GGCXCC-3', where X = C8-dG adduct), however there is still limited capacity to predict the likelihood of mutation arising within particular contexts, and hence chemistry-based strategies are needed for probing relationships between nucleic acid sequence and structure with replication errors. In the NarI sequence, certain C8-dG adducts may trigger in the course of DNA synthesis the formation of a slipped mutagenic intermediate (SMI) that contains a two nucleotide (XC) bulge in the template strand that can form upstream of the polymerase active site. This distortion facilitates polymerization but affords a GC dinucleotide deletion product (-2 frameshift mutation). In the current study, incorporating the fluorescent C-linked 4-fluorobiphenyl-dG (FBP-dG) adduct into two 22-mer templates containing CG-dinucleotide repeats ( NarI: 3'-CXCGGC-5' and CG3: 3'-CXCGCG-5', X = FBP-dG) and performing primer extension reactions using DNA polymerase I, Klenow fragment exo- (Kf-) revealed a dramatic sequence-based difference in polymerase bypass efficiency. Primer extension past FBP-dG within the NarI sequence was strongly blocked, whereas Kf- extended the primer past FBP-dG within a CG3 template to afford a full-length product and the GC dinucleotide deletion. To model the nucleotide insertion steps in the fully paired (FP) versus the slipped mutagenic (SM) translesion pathways, adducted template:primer duplexes were constructed and characterized by UV thermal denaturation and fluorescence spectroscopy. The emission intensity of the FBP-dG lesion exhibits sensitivity to SMI formation (turn-on) versus a FP duplex (turn-off), permitting insight into adduct base-pairing within the template:primer duplexes. This fluorescence sensitivity provides a rationale for sequence impact on -2 frameshift mutations mediated by the C-linked FBP-dG lesion.