A cloud-based learning module for biomarker discovery.

This manuscript describes the development of a resource module that is part of a learning platform named "NIGMS Sandbox for Cloud-based Learning" https://github.com/NIGMS/NIGMS-Sandbox. The overall genesis of the Sandbox is described in the editorial NIGMS Sandbox at the beginning of this Supplement. This module delivers learning materials on basic principles in biomarker discovery in an interactive format that uses appropriate cloud resources for data access and analyses. In collaboration with Google Cloud, Deloitte Consulting and NIGMS, the Rhode Island INBRE Molecular Informatics Core developed a cloud-based training module for biomarker discovery. The module consists of nine submodules covering various topics on biomarker discovery and assessment and is deployed on the Google Cloud Platform and available for public use through the NIGMS Sandbox. The submodules are written as a series of Jupyter Notebooks utilizing R and Bioconductor for biomarker and omics data analysis. The submodules cover the following topics: 1) introduction to biomarkers; 2) introduction to R data structures; 3) introduction to linear models; 4) introduction to exploratory analysis; 5) rat renal ischemia-reperfusion injury case study; (6) linear and logistic regression for comparison of quantitative biomarkers; 7) exploratory analysis of proteomics IRI data; 8) identification of IRI biomarkers from proteomic data; and 9) machine learning methods for biomarker discovery. Each notebook includes an in-line quiz for self-assessment on the submodule topic and an overview video is available on YouTube (https://www.youtube.com/watch?v=2-Q9Ax8EW84). This manuscript describes the development of a resource module that is part of a learning platform named ``NIGMS Sandbox for Cloud-based Learning'' https://github.com/NIGMS/NIGMS-Sandbox. The overall genesis of the Sandbox is described in the editorial NIGMS Sandbox [1] at the beginning of this Supplement. This module delivers learning materials on the analysis of bulk and single-cell ATAC-seq data in an interactive format that uses appropriate cloud resources for data access and analyses.

Conformation-dependent lesion bypass of bulky arylamine-dG adducts generated from 2-nitrofluorene in epigenetic sequence contexts.

Sequence context influences structural characteristics and repair of DNA adducts, but there is limited information on how epigenetic modulation affects conformational heterogeneity and bypass of DNA lesions. Lesions derived from the environmental pollutant 2-nitrofluorene have been extensively studied as chemical carcinogenesis models; they adopt a sequence-dependent mix of two significant conformers: major groove binding (B) and base-displaced stacked (S). We report a conformation-dependent bypass of the N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene (dG-FAF) lesion in epigenetic sequence contexts (d[5'-CTTCTC#G*NCCTCATTC-3'], where C# is C or 5-methylcytosine (5mC), G* is G or G-FAF, and N is A, T, C or G). FAF-modified sequences with a 3' flanking pyrimidine were better bypassed when the 5' base was 5mC, whereas sequences with a 3' purine exhibited the opposite effect. The conformational basis behind these variations differed; for -CG*C- and -CG*T-, bypass appeared to be inversely correlated with population of the duplex-destabilizing S conformer. On the other hand, the connection between conformation and a decrease in bypass for flanking purines in the 5mC sequences relative to C was more complex. It could be related to the emergence of a disruptive non-S/B conformation. The present work provides novel conformational insight into how 5mC influences the bypass efficiency of bulky DNA damage.

Probing the Interactions of Perfluorocarboxylic Acids of Various Chain Lengths with Human Serum Albumin: Calorimetric and Spectroscopic Investigations.

Despite an exponential increase in PFAS research over the past two decades, the mechanisms behind how PFAS cause adverse health effects are still poorly understood. Protein interactions are considered a significant driver of bioaccumulation and subsequent toxicity from re-exposure; however, most of the available literature is limited to legacy PFAS. We utilized microcalorimetric and spectroscopic methods to systematically investigate the binding between human serum albumin (HSA) and perfluorocarboxylic acids (PFCAs) of varying chain lengths and their nonfluorinated fatty acid (FA) counterparts. The results reveal the optimal chain length for significant PFCA-HSA binding and some fundamental interactions, i.e., the polar carboxylic head of PFCA is countered by ionizable amino acids such as arginine, and the fluorocarbon tails stabilized by hydrophobic residues like leucine and valine. Additionally, fluorine's unique polarizability contributes to PFCA's stronger binding affinities relative to the corresponding fatty acids. Based on these observations, we posit that PFCAs likely bind to HSA in a "cavity-filling" manner, provided they have an appropriate size and shape to accommodate the electrostatic interactions. The results reported herein widen the pool of structural information to explain PFAS bioaccumulation patterns and toxicity and support the development of more accurate computational modeling of protein-PFAS interactions. TOC graphic created with Biorender.com.

DNA base sequence effects on bulky lesion-induced conformational heterogeneity during DNA replication.

4-Aminobiphenyl (ABP) and its structure analog 2-aminofluorene (AF) are well-known carcinogens. In the present work, an unusual sequence effect in the 5'-CTTCTG1G2TCCTCATTC-3' DNA duplex is reported for ABP- and AF-modified G. Specifically, the ABP modification at G1 resulted in a mixture of 67% major groove B-type (B) and 33% stacked (S) conformers, while at the ABP modification at G2 exclusively resulted in the B-conformer. The AF modification at G1 and G2 lead to 25%:75% and 83%:17% B:S population ratios, respectively. These differences in preferred conformation are due to an interplay between stabilizing (hydrogen bonding and stacking that is enhanced by lesion planarity) and destabilizing (solvent exposure) forces at the lesion site. Furthermore, while the B-conformer is a thermodynamic stabilizer and the S-conformer is a destabilizer in duplex settings, the situation is reversed at the single strands/double strands (ss/ds) junction. Specifically, the twisted biphenyl is a better stacker at the ss/ds junction than the coplanar AF. Therefore, the ABP modification leads to a stronger strand binding affinity of the ss/ds junction than the AF modification. Overall, the current work provides conformational insights into the role of sequence and lesion effects in modulating DNA replication.

Probing the Effect of Bulky Lesion-Induced Replication Fork Conformational Heterogeneity Using 4-Aminobiphenyl-Modified DNA.

Bulky organic carcinogens are activated in vivo and subsequently react with nucleobases of cellular DNA to produce adducts. Some of these DNA adducts exist in multiple conformations that are slowly interconverted to one another. Different conformations have been implicated in different mutagenic and repair outcomes. However, studies on the conformation-specific inhibition of replication, which is more relevant to cell survival, are scarce, presumably due to the structural dynamics of DNA lesions at the replication fork. It is difficult to capture the exact nature of replication inhibition by existing end-point assays, which usually detect either the ensemble of consequences of all the conformers or the culmination of all cellular behaviors, such as mutagenicity or survival rate. We previously reported very unusual sequence-dependent conformational heterogeneities involving FABP-modified DNA under different sequence contexts (TG1*G2T [67%B:33%S] and TG1G2*T [100%B], G*, N-(2'-deoxyguanosin-8-yl)-4'-fluoro-4-aminobiphenyl) (Cai et al. Nucleic Acids Research, 46, 6356-6370 (2018)). In the present study, we attempted to correlate the in vitro inhibition of polymerase activity to different conformations from a single FABP-modified DNA lesion. We utilized a combination of surface plasmon resonance (SPR) and HPLC-based steady-state kinetics to reveal the differences in terms of binding affinity and inhibition with polymerase between these two conformers (67%B:33%S and 100%B).

Conformational Insights into the Mechanism of Acetylaminofluorene-dG-Induced Frameshift Mutations in the NarI Mutational Hotspot.

Frameshift mutagenesis encompasses the gain or loss of DNA base pairs, resulting in altered genetic outcomes. The NarI restriction site sequence 5'-G1G2CG3CX-3' in Escherichia coli is a well-known mutational hotspot, in which lesioning of acetylaminofluorene (AAF) at G3* induces a greater -2 deletion frequency than that at other guanine sites. Its mutational efficiency is modulated by the nature of the nucleotide in the X position (C ∼ A > G ≫ T). Here, we conducted a series of polymerase-free solution experiments that examine the conformational and thermodynamic basis underlying the propensity of adducted G3 to form a slipped mutagenic intermediate (SMI) and its sequence dependence during translesion synthesis (TLS). Instability of the AAF-dG3:dC pair at the replication fork promoted slippage to form a G*C bulge-out SMI structure, consisting of S- ("lesion stacked") and B-SMI ("lesion exposed") conformations, with conformational rigidity increasing as a function of primer elongation. We found greater stability of the S- compared to the B-SMI conformer throughout TLS. The dependence of their population ratios was determined by the 3'-next flanking base X at fully elongated bulge structures, with 59% B/41% S and 86% B/14% S for the dC and dT series, respectively. These results indicate the importance of direct interactions of the hydrophobic AAF lesion with the 3'-next flanking base pair and its stacking fit within the -2 bulge structure. A detailed conformational understanding of the SMI structures and their sequence dependence may provide a useful model for DNA polymerase complexes.

DNA-binding studies of the natural ß-carboline eudistomin U.

Eudistomin U is a member of the ß-carboline class of heterocyclic amine-containing molecules that are capable of binding to DNA. The structure of eudistomin U is unique since it contains an indole ring at the 1-position of the pyridine ring. While simple ß-carbolines are reported to intercalate DNA, an examination of the mode of binding of eudistomin U has been lacking. We report preliminary spectroscopic (UV-Vis, thermal denaturation, CD) and calorimetric (DSC) data on the binding of eudistomin U to DNA, which suggest that eudistomin U binds weakly according to a mechanism that is more complicated than other members of its class.

Unusual sequence effects on nucleotide excision repair of arylamine lesions: DNA bending/distortion as a primary recognition factor.

The environmental arylamine mutagens are implicated in the etiology of various sporadic human cancers. Arylamine-modified dG lesions were studied in two fully paired 11-mer duplexes with a -G*CN- sequence context, in which G* is a C8-substituted dG adduct derived from fluorinated analogs of 4-aminobiphenyl (FABP), 2-aminofluorene (FAF) or 2-acetylaminofluorene (FAAF), and N is either dA or dT. The FABP and FAF lesions exist in a simple mixture of 'stacked' (S) and 'B-type' (B) conformers, whereas the N-acetylated FAAF also samples a 'wedge' (W) conformer. FAAF is repaired three to four times more efficiently than FABP and FAF. A simple A- to -T polarity swap in the G*CA/G*CT transition produced a dramatic increase in syn-conformation and resulted in 2- to 3-fold lower nucleotide excision repair (NER) efficiencies in Escherichia coli. These results indicate that lesion-induced DNA bending/thermodynamic destabilization is an important DNA damage recognition factor, more so than the local S/B-conformational heterogeneity that was observed previously for FAF and FAAF in certain sequence contexts. This work represents a novel 3'-next flanking sequence effect as a unique NER factor for bulky arylamine lesions in E. coli.

Conformational insights into the lesion and sequence effects for arylamine-induced translesion DNA synthesis: 19F NMR, surface plasmon resonance, and primer kinetic studies.

Adduct-induced DNA damage can affect transcription efficiency and DNA replication and repair. We previously investigated the effects of the 3'-next flanking base (G*CT vs G*CA; G*, FABP, N-(2'-deoxyguanosin-8-yl)-4'-fluoro-4-aminobiphenyl; FAF, N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene) on the conformation of arylamine-DNA lesions in relation to E. coli nucleotide excision repair ( Jain , V. , Hilton , B. , Lin , B. , Patnaik , S. , Liang , F. , Darian , E. , Zou , Y. , Mackerell , A. D. , Jr. , and Cho , B. P. ( 2013 ) Nucleic Acids Res. , 41 , 869 - 880 ). Here, we report the differential effects of the same pair of sequences on DNA replication in vitro by the polymerases exofree Klenow fragment (Kf-exo(-)) and Dpo4. We obtained dynamic (19)F NMR spectra for two 19-mer modified templates during primer elongation: G*CA [d(5'-CTTACCATCG*CAACCATTC-3')] and G*CT [d(5'-CTTACCATCG*CTACCATTC-3')]. We found that lesion stacking is favored in the G*CT sequence compared to the G*CA counterpart. Surface plasmon resonance binding results showed consistently weaker affinities for the modified DNA with the binding strength in the order of FABP > FAF and G*CA > G*CT. Primer extension was stalled at (n) and near (n - 1 and n + 1) the lesion site, and the extent of blockage and the extension rates across the lesion were influenced by not only the DNA sequences but also the nature of the adduct's chemical structure (FAF vs FABP) and the polymerase employed (Kf-exo(-) vs Dpo4). Steady-state kinetics analysis with Kf-exo(-) revealed the most dramatic sequence and lesion effects at the lesion (n) and postinsertion (n + 1) sites, respectively. Taken together, these results provide insights into the important role of lesion-induced conformational heterogeneity in modulating translesion DNA synthesis.

Insights into the conformation of aminofluorene-deoxyguanine adduct in a DNA polymerase active site.

The active site conformation of the mutagenic fluoroaminofluorene-deoxyguanine adduct (dG-FAF, N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene) has been investigated in the presence of Klenow fragment of Escherichia coli DNA polymerase I (Kfexo(-)) and DNA polymerase ß (pol ß) using (19)F NMR, insertion assay, and surface plasmon resonance. In a single nucleotide gap, the dG-FAF adduct adopts both a major-groove- oriented and base-displaced stacked conformation, and this heterogeneity is retained upon binding pol ß. The addition of a non-hydrolysable 2'-deoxycytosine-5'-[(α,ß)-methyleno]triphosphate (dCMPcPP) nucleotide analog to the binary complex results in an increase of the major groove conformation of the adduct at the expense of the stacked conformation. Similar results were obtained with the addition of an incorrect dAMPcPP analog but with formation of the minor groove binding conformer. In contrast, dG-FAF adduct at the replication fork for the Kfexo(-) complex adopts a mix of the major and minor groove conformers with minimal effect upon the addition of non-hydrolysable nucleotides. For pol ß, the insertion of dCTP was preferred opposite the dG-FAF adduct in a single nucleotide gap assay consistent with (19)F NMR data. Surface plasmon resonance binding kinetics revealed that pol ß binds tightly with DNA in the presence of correct dCTP, but the adduct weakens binding with no nucleotide specificity. These results provide molecular insights into the DNA binding characteristics of FAF in the active site of DNA polymerases and the role of DNA structure and sequence on its coding potential.

Real-time surface plasmon resonance study of biomolecular interactions between polymerase and bulky mutagenic DNA lesions.

Surface plasmon resonance (SPR) was used to measure polymerase-binding interactions of the bulky mutagenic DNA lesions N-(2'-deoxyguanosin-8-yl)-4'-fluoro-4-aminobiphenyl (FABP) or N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-acetylaminofluorene (FAAF) in the context of two unique 5'-flanking bases (CG*A and TG*A). The enzymes used were exo-nuclease-deficient Klenow fragment (Kf-exo(-)) or polymerase ß (pol ß). Specific binary and ternary DNA binding affinities of the enzymes were characterized at subnanomolar concentrations. The SPR results showed that Kf-exo(-) binds strongly to a double strand/single strand template/primer junction, whereas pol ß binds preferentially to double-stranded DNA having a one-nucleotide gap. Both enzymes exhibited tight binding to native DNA, with high nucleotide selectivity, where the KD values for each base pair increased in the order dCTP ≪ dTTP ∼ dATP ≪ dGTP. In contrast to that for pol ß, Kf-exo(-) binds tightly to lesion-modified templates; however, both polymerases exhibited minimal nucleotide selectivity toward adducted DNA. Primer steady-state kinetics and (19)F NMR results support the SPR data. The relative insertion efficiency fins of dCTP opposite FABP was significantly higher in the TG*A sequence compared to that in CG*A. Although Kf-exo(-) was not sensitive to the presence of a DNA lesion, FAAF-induced conformational heterogeneity perturbed the active site of pol ß, weakening the enzyme's ability to bind to FAAF adducts compared to FABP adducts. The present study demonstrates the effectiveness of SPR for elucidating how lesion-induced conformational heterogeneity affects the binding capability of polymerases and ultimately the nucleotide insertion efficiency.

Conformational and thermodynamic properties modulate the nucleotide excision repair of 2-aminofluorene and 2-acetylaminofluorene dG adducts in the NarI sequence.

Nucleotide excision repair (NER) is a major repair pathway that recognizes and corrects various lesions in cellular DNA. We hypothesize that damage recognition is an initial step in NER that senses conformational anomalies in the DNA caused by lesions. We prepared three DNA duplexes containing the carcinogen adduct N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-acetylaminofluorene (FAAF) at G(1), G(2) or G(3) of NarI sequence (5'-CCG(1)G(2)CG(3)CC-3'). Our (19)F-NMR/ICD results showed that FAAF at G(1) and G(3) prefer syn S- and W-conformers, whereas anti B-conformer was predominant for G(2). We found that the repair of FAAF occurs in a conformation-specific manner, i.e. the highly S/W-conformeric G(3) and -G(1) duplexes incised more efficiently than the B-type G(2) duplex (G(3)∼G(1)> G(2)). The melting and thermodynamic data indicate that the S- and W-conformers produce greater DNA distortion and thermodynamic destabilization. The N-deacetylated N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene (FAF) adducts in the same NarI sequence are repaired 2- to 3-fold less than FAAF: however, the incision efficiency was in order of G(2)∼G(1)> G(3), a reverse trend of the FAAF case. We have envisioned the so-called N-acetyl factor as it could raise conformational barriers of FAAF versus FAF. The present results provide valuable conformational insight into the sequence-dependent UvrABC incisions of the bulky aminofluorene DNA adducts.

Structure and thermodynamic insights on acetylaminofluorene-modified deletion DNA duplexes as models for frameshift mutagenesis.

2-Acetylaminofluorene (AAF) is a prototype arylamine carcinogen that forms C8-substituted dG-AAF and dG-AF as the major DNA lesions. The bulky N-acetylated dG-AAF lesion can induce various frameshift mutations depending on the base sequence around the lesion. We hypothesized that the thermodynamic stability of bulged-out slipped mutagenic intermediates (SMIs) is directly related to deletion mutations. The objective of the present study was to probe the structural/conformational basis of various dG-AAF-induced SMIs formed during translesion synthesis. We performed spectroscopic, thermodynamic, and molecular dynamics studies of several AAF-modified 16-mer model DNA duplexes, including fully paired and -1, -2, and -3 deletion duplexes of the 5'-CTCTCGATG[FAAF]CCATCAC-3' sequence and an additional -1 deletion duplex of the 5'-CTCTCGGCG[FAAF]CCATCAC-3' NarI sequence. Modified deletion duplexes existed in a mixture of external B and stacked S conformers, with the population of the S conformer being 'GC'-1 (73%) > 'AT'-1 (72%) > full (60%) > -2 (55%) > -3 (37%). Thermodynamic stability was in the order of -1 deletion > -2 deletion > fully paired > -3 deletion duplexes. These results indicate that the stacked S-type conformer of SMIs is thermodynamically more stable than the conformationally flexible external B conformer. Results from the molecular dynamics simulations indicate that perturbation of base stacking dominates the relative stability along with contributions from bending, duplex dynamics, and solvation effects that are important in specific cases. Taken together, these results support a hypothesis that the conformational and thermodynamic stabilities of the SMIs are critical determinants for the induction of frameshift mutations.

Structural and thermodynamic insight into Escherichia coli UvrABC-mediated incision of cluster diacetylaminofluorene adducts on the NarI sequence.

Cluster DNA damage refers to two or more lesions in a single turn of the DNA helix. Such clustering may occur with bulky DNA lesions, which may be responsible for their sequence-dependent repair and mutational outcomes. Here we prepared three 16-mer cluster duplexes in which two fluoroacetylaminofluorene adducts (dG-FAAF) are separated by zero, one, and two nucleotides in the Escherichia coli NarI mutational hot spot (5'-CTCTCG1G2CG3CCATCAC-3'): 5'-CG1*G2*CG3CC-3', 5'-CG1G2*CG3*CC-3', and 5'-CG1*G2CG3*CC-3' (G* = dG-FAAF), respectively. We conducted spectroscopic, thermodynamic, and molecular dynamics studies of these di-FAAF duplexes, and the results were compared with those of the corresponding mono-FAAF adducts in the same NarI sequence [Jain, V., et al. (2012) Nucleic Acids Res. 40, 3939-3951]. Our nucleotide excision repair results showed the diadducts were more reparable than the corresponding monoadducts. Moreover, we observed dramatic flanking base sequence effects on their repair efficiency in the following order: NarI-G2G3 > NarI-G1G3 > NarI-G1G2. The nuclear magnetic resonance, circular dichroism, ultraviolet melting, and molecular dynamics simulation results revealed that in contrast to the monoadducts, diadducts produced a synergistic effect on duplex destabilization. In addition, dG-FAAF at G2G3 and G1G3 destacks the neighboring bases, with greater destabilization occurring with the former. Overall, the results indicate the importance of base stacking and related thermal and thermodynamic destabilization in the repair of bulky cluster arylamine DNA adducts.

Developing virtual and augmented reality applications for science, technology, engineering and math education.

The Rhode Island IDeA Network of Biomedical Research Excellence Molecular Informatics Core at the University of Rhode Island Information Technology Services Innovative Learning Technologies developed virtual and augmented reality applications to teach concepts in biomedical science, including pharmacology, medicinal chemistry, cell culture and nanotechnology. The apps were developed as full virtual reality/augmented reality and 3D gaming versions, which do not require virtual reality headsets. Development challenges included creating intuitive user interfaces, text-to-voice functionality, visualization of molecules and implementing complex science concepts. In-app quizzes are used to assess the user's understanding of topics, and user feedback was collected for several apps to improve the experience. The apps were positively reviewed by users and are being implemented into the curriculum at the University of Rhode Island.

Sequence effects on translesion synthesis of an aminofluorene-DNA adduct: conformational, thermodynamic, and primer extension kinetic studies.

The DNA sequence effect is an important structural factor for determining the extent and nature of carcinogen-induced mutational and repair outcomes. In this study, we used two 16-mer template sequences, TG*A [d(5'-CTTCTTG*ACCTCATTC-3')] and CG*A [d(5'-CTTCTCG*ACCTCATTC-3')], to study the impact of the 5'-flanking nucleotide (T vs C) on aminofluorene (AF)-induced stacked (S)/major groove (B)/wedge (W) conformational heterogeneity during a simulated translesion synthesis. In addition, we probed the sequence effect on nucleotide insertion efficiencies catalyzed by the Klenow fragment (exonuclease-deficient) of DNA polymerase I. Our (19)F NMR/ICD/DSC results showed that AF in the CG*A duplex sequence adopts a greater population of S-conformer than the TG*A sequence. We found that the S conformer of CG*A thermodynamically favors insertion of A over C at the lesion site (n). Significant stalling occurred at both the prelesion (n - 1) and lesion (n) sites; however, the effect was more persistent for the S conformer of CG*A than TG*A at the lesion site (n). Kinetics show that relative nucleotide insertion frequencies (f(ins)) were greater for TG*A than the S conformer of CG*A for either dCTP or dATP at the lesion site (n), and the insertion rate was significantly reduced at immediate upstream base pairs (n, n + 1). Taken together, the results provide insight into how the mutagenic AF could exhibit an S/B/W equilibrium in the active site of a polymerase, causing different mutations. This work represents a novel structure-function relationship in which adduct structure is directly linked to nucleotide insertion efficiency in a conformation-specific manner during translesion DNA synthesis.

Binary and ternary binding affinities between exonuclease-deficient Klenow fragment (Kf-exo(-)) and various arylamine DNA lesions characterized by surface plasmon resonance.

We used surface plasmon resonance (SPR) to characterize the binding interactions between the exonulease-free Klenow fragment (Kf-exo(-)) and unmodified and modified dG adducts derived from arylamine carcinogens: fluorinated 2-aminofluorene (FAF), 2-acetylaminofluorene (FAAF), and 4-aminobiphenyl (FABP). Tight polymerase binding was detected with unmodified dG and the correct dCTP. The discrimination of correct versus incorrect nucleotides was pronounced with K(D) values in the order of dCTP ≪ dTTP < dATP < dGTP. In contrast, minimal selectivity was observed for the modified templates with Kf-exo(-) binding tighter to the FAAF (k(off): 0.02 s(-1)) and FABP (k(off): 0.01 s(-1)) lesions than to FAF (k(off): 0.04 s(-1)).

Characterization of molecular interactions between cannabidiol and human plasma proteins (serum albumin and γ-globulin) by surface plasmon resonance, microcalorimetry, and molecular docking.

A cannabidiol (CBD) oral solution (Epidiolex®) has been approved by the United States Food and Drug Administration to treat seizure conditions. However, the biomedical and pharmaceutical applications of CBD are hindered partially due to a limited understanding of CBD's pharmacokinetic behaviors, such as its interactions with plasma proteins. Herein, we investigated the molecular interactions between CBD and two plasma proteins, namely, human serum albumin (HSA) and γ-globulin, using biophysical techniques including surface plasmon resonance (SPR), isothermal titration calorimetry, and differential scanning calorimetry, as well as molecular docking. CBD bound to HSA and γ-globulin in an exothermic manner (enthalpy: -9.3 ×104 and -3.7 ×104 kcal/mol, respectively) with a binding affinity of 1.8 × 10-5 and 1.3 × 10-5 M, respectively. The binding ratio between CBD and HSA or γ-globulin was approximately 1:1 and 3:1, respectively. Furthermore, computational modeling suggested that CBD and warfarin may bind to HSA independently, supported by data from a competitive SPR binding assay. Findings from the current study elucidate CBD's plasma protein binding characteristics and shed light on their impact on CBD's pharmacokinetic property.

Conformational and thermodynamic impact of bulky aminofluorene adduction on simulated translesion DNA synthesis.

We report a systematic spectroscopic investigation on the conformational evolution during primer extension of a bulky fluoroaminofluorene-modified dG adduct (FAF-dG) in chemically simulated translesion synthesis. FAF-dG was paired either with dC or dA (dC-match and dA-mismatch series, respectively). Dynamic (19)F NMR/CD results showed that the FAF-adduct exists in a syn/anti equilibrium and that its conformational characteristics are modulated by the identity of an inserted nucleotide at the lesion site and the extent of primer elongation. At the pre-insertion site, the adduct adopted preferentially a syn conformation where FAF stacked with preceding bases. Insertion of the correct nucleotide dC at the lesion site and subsequent elongation resulted in a gradual transition to the anti conformation. By contrast, the syn conformer was persistent along with primer extension in the dA-mismatch series. In the dC-match series, FAF-induced thermal (T(m)) and thermodynamic (-ΔG°(37 °C)) stabilities were significantly reduced relative to those of the controls. However, the corresponding T(m) and -ΔG°(37 °C) values were increased in the FAF-modified mismatched dA series. The lesion impact persisted up to three 5'-nucleotides from the lesion. Occupation of the minor groove of the W-conformer with the bulky carcinogenic fluorene moiety not only would limit the DNA mobility but also would impose a serious difficulty for the active site of a polymerase throughout the replication process. Our spectroscopic results are consistent with reported data on AF, which showed dramatic (~10(4)-fold) differences in the nucleotide insertion rates between the dC-match and dA-mismatch series. The results emphasize the importance of adduct-induced steric constraints for determining the replication fidelity of a polymerase.

Influence of flanking sequence context on the conformational flexibility of aminofluorene-modified dG adduct in dA mismatch DNA duplexes.

When positioned opposite to a dA in a DNA duplex, the prototype arylamine-DNA adduct [N-(2'-deoxyguanosin-yl)-7-fluoro-2-aminofluorene (FAF)] adopts the so-called 'wedge' (W) conformation, in which the carcinogen resides in the minor groove of the duplex. All 16 FAF-modified 12-mer NG*N/NAN dA mismatch duplexes (G* = FAF, N = G, A, C, T) exhibited strongly positive induced circular dichroism in the 290-360 nm range (ICD(290-360 nm)), which supports the W conformation. The ICD(290-360 nm) intensities were the greatest for duplexes with a 3'-flanking T. The AG*N duplex series showed little adduct-induced destabilization. An exception was the AG*T duplex, which displayed two well-resolved signals in the (19)F NMR spectra. This was presumably due to a strong lesion-destabilizing effect of the 3'-T. The flanking T effect was substantiated further by findings with the TG*T duplex, which exhibited greater lesion flexibility and nucleotide excision repair recognition. Adduct conformational heterogeneity decreased in order of TG*T > AG*T > CG*T > AG*A > AG*G > AG*C. The dramatic flanking T effect on W-conformeric duplexes is consistent with the strong dependence of the ICD(290-360) on both temperature and salt concentration and could be extended to the arylamine food mutagens that are biologically relevant in humans.