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B-form DNA can adopt alternative structures under conditions such as superhelical duress. Alternative DNA structures are favored when there is asymmetric distribution of guanosine and cytosine on complimentary DNA strands. A guanosine-rich strand can form a four-stranded structure known as a quadruplex (G4). The complimentary cytosine-rich strand can utilize intercalating cytosine–cytosine base pairing to form a four-stranded structure known as the i-motif (iM). Both secondary structures are energetically uphill from double-strand DNA (dsDNA), meaning that additional factors are needed for their formation. Most iMs require slightly acidic conditions for structure stabilization. However, crowding agents such as polyethylene glycols and dextrans can shift the pKa of the iM to near-physiological pH ≈ 7. Nucleic acids have long been known to be bound and stabilized by polyamines such as putrescine, spermidine, and spermine. Polyamines have very high concentrations in cells (0.1–30 mM), and their binding to DNA is driven by electrostatic interactions. Polyamines typically bind in the minor groove of DNA. However, because of the unusual structure of iMs, it was unknown whether polyamines might also bind and stabilize iMs. The study described here was undertaken to analyze polyamine–iM interactions. The thermal stability and pH dependence of iM structures were determined in the presence of polyamines. In contrast to dsDNA, our results suggest that polyamines have considerably weaker interactions with iMs, as demonstrated by the minimal change in iM pH dependence and thermal stability. Our results suggest that polyamines are unlikely to provide a significant source of iM stabilization in vivo.

G-quadruplexes (G4s) and i-motifs (iMs) are tetraplex DNA structures. Sequences capable of forming G4/iMs are abundant near the transcription start sites (TSS) of several genes. G4/iMs affect gene expression in vitro. Depending on the gene, the presence of G4/iMs can enhance or suppress expression, making it challenging to discern the underlying mechanism by which they operate. Factors affecting G4/iM structures can provide additional insight into their mechanism of regulation. One such factor is epigenetic modification. The 5-hydroxymethylated cytosines (5hmCs) are epigenetic modifications that occur abundantly in human embryonic stem cells (hESC). The 5hmCs, like G4/iMs, are known to participate in gene regulation and are also enriched near the TSS. We investigated genomic co-localization to assess the possibility that these two elements may play an interdependent role in regulating genes in hESC. Our results indicate that amongst 15,760 G4/iM-forming locations, only 15% have 5hmCs associated with them. A detailed analysis of G4/iM-forming locations enriched in 5hmC indicates that most of these locations are in genes that are associated with cell differentiation, proliferation, apoptosis and embryogenesis. The library generated from our analysis is an important resource for investigators exploring the interdependence of these DNA features in regulating expression of selected genes in hESC.

The four-stranded i-motif (iM) conformation of cytosine-rich DNA is important in a wide variety of biochemical systems ranging from its use in nanomaterials to a potential role in oncogene regulation. An iM is stabilized by acidic pH that allows hemiprotonated cytidines to form a C·C(+) base pair. Fundamental studies that aim to understand how the lengths of loops connecting the protonated C·C(+) pairs affect intramolecular iM physical properties are described here. We characterized both the thermal stability and the pK(a) of intramolecular iMs with differing loop lengths, in both dilute solutions and solutions containing molecular crowding agents. Our results showed that intramolecular iMs with longer central loops form at pHs and temperatures higher than those of iMs with longer outer loops. Our studies also showed that increases in thermal stability of iMs when molecular crowding agents are present are dependent on the loop that is lengthened. However, the increase in pK(a) for iMs when molecular crowding agents are present is insensitive to loop length. Importantly, we also determined the proton activity of solutions containing high concentrations of molecular crowding agents to ascertain whether the increase in pK(a) of an iM is caused by alteration of this activity in buffered solutions. We determined that crowding agents alone increase the apparent pK(a) of a number of small molecules as well as iMs but that increases to iM pK(a) were greater than that expected from a shift in proton activity.

Promoters often contain asymmetric G- and C-rich strands, in which the cytosines are prone to epigenetic modification via methylation (5-mC) and 5-hydroxymethylation (5-hmC). These sequences can also form four-stranded G-quadruplex (G4) or i-motif (iM) secondary structures. Although the requisite sequences for epigenetic modulation and iM/G4 formation are similar and can overlap, they are unlikely to coexist. Despite 5-hmC being an oxidization product of 5-mC, the two modified bases cluster at distinct loci. This study focuses on the intersection of G4/iM formation and 5-hmC modification using the vascular endothelial growth factor (VEGF) gene promoter’s CpG sites and examines whether incorporation of 5-hmC into iM/G4 structures had any physicochemical effect on formation, stability, or recognition by nucleolin or the cationic porphyrin, TMPyP4. No marked changes were found in the formation or stability of iM and G4 structures; however, changes in recognition by nucleolin or TMPyP4 occurred with 5-hmC modification wherein protein and compound binding to 5-hmC modified G4s was notably reduced. G4/iM structures in the VEGF promoter are promising therapeutic targets for antiangiogenic therapy, and this work contributes to a comprehensive understanding of their governing principles related to potential transcriptional control and targeting.

Fisetin (3,7,3',4'-tetrahydroxyflavone) and quercetin (3,5,7,3',4'-pentahydroxyflavone) are the bioactive plant flavonoids that are potentially useful therapeutic drugs for the treatment of a broad spectrum of diseases, including atherosclerosis, cardiovascular disease, obesity, hypertension, and cancer. 3-Hydroxyflavone (3HF) and 7-hydroxyflavone (7HF) are the synthetic chromophores of fisetin and quercetin. We have exploited dual luminescence properties of fisetin and quercetin along with 3-HF and 7HF to examine their efficacy of binding and compare their interactions with DNA, which is one of the macromolecular targets of flavonoids in physiological systems. Following the sequence of the human telomeric DNA 5'-d (CCCTAA-)n/(-TTAGGG)n-5', two single-stranded DNA oligonucleotides, 5'-d(C3TA2)3C3-3' and 5'-d(T2AG3)4-3', and their duplex were used as receptors to study binding by the ligands quercetin, fisetin, and their chromophores. Circular dichroism, differential absorption, UV thermal melting, and size exclusion chromatographic studies indicated the formation of unusual DNA structures (such as C4 and G4 tetraplexes) for both the C- and G-rich single-stranded DNAs. Upon binding to DNA, dramatic changes were observed in the intrinsic fluorescence behavior of the flavonoids. Molecular docking studies were performed to describe the likely binding sites for the ligands. The spectroscopic studies on flavonoid-DNA interactions described herein demonstrate a powerful approach for examining their DNA binding through exploiting the highly sensitive intrinsic fluorescence properties of the flavonoids as their own "reporter" for their interactions with macromolecular targets.

The four-stranded i-motif (iM) conformation of cytosine-rich DNA has importance to a wide variety of biochemical systems that range from their use in nanomaterials to potential roles in oncogene regulation. The iM structure is formed at slightly acidic pH, where hemiprotonation of cytosine results in a stable C-C+ basepair. Here, we performed fundamental studies to examine iM formation from a C-rich strand from the promoter of the human c-MYC gene. We used a number of biophysical techniques to characterize both the hydrodynamic properties and folding kinetics of a folded iM. Our hydrodynamic studies using fluorescence anisotropy decay and analytical ultracentrifugation show that the iM structure has a compact size in solution and displays the rigidity of a double strand. By studying the rates of circular dichroism spectral changes and quenching of fluorescent cytidine analogs, we also established a mechanism for the folding of a random coil oligo into the iM. In the course of determining this folding pathway, we established that the fluorescent dC analogs tC° and PdC can be used to monitor individual residues of an iM structure and to determine the pKa of an iM. We established that the C-C+ hydrogen bonding of certain bases initiates the folding of the iM structure. We also showed that substitutions in the loop regions of iMs give a distinctly different kinetic signature during folding compared with bases that are intercalated. Our data reveal that the iM passes through a distinct intermediate form between the unfolded and folded forms. Taken together, our results lay the foundation for using fluorescent dC analogs to follow structural changes during iM formation. Our technique may also be useful for examining folding and structural changes in more complex iMs.

DNA sequences with the potential to form secondary structures such as i-motifs (iMs) and G-quadruplexes (G4s) are abundant in the promoters of several oncogenes and, in some instances, are known to regulate gene expression. Recently, iM-forming DNA strands have also been employed as functional units in nanodevices, ranging from drug delivery systems to nanocircuitry. To understand both the mechanism of gene regulation by iMs and how to use them more efficiently in nanotechnological applications, it is essential to have a thorough knowledge of factors that govern their conformational states and stabilities. Most of the prior work to characterize the conformational dynamics of iMs have been done with iM-forming synthetic constructs like tandem (CCT)n repeats and in standard dilute buffer systems. Here, we present a systematic study on the consequences of epigenetic modifications, molecular crowding, and degree of hydration on the stabilities of an iM-forming sequence from the promoter of the c-myc gene. Our results indicate that 5-hydroxymethylation of cytosines destabilized the iMs against thermal and pH-dependent melting; contrarily, 5-methylcytosine modification stabilized the iMs. Under molecular crowding conditions (PEG-300, 40% w/v), the thermal stability of iMs increased by ∼10 °C, and the pKa was raised from 6.1 ± 0.1 to 7.0 ± 0.1. Lastly, the iM's stability at varying degrees of hydration in 1,2-dimethoxyethane, 2-methoxyethanol, ethylene glycol, 1,3-propanediol, and glycerol cosolvents indicated that the iMs are stabilized by dehydration because of the release of water molecules when folded. Our results highlight the importance of considering the effects of epigenetic modifications, molecular crowding, and the degree of hydration on iM structural dynamics. For example, the incorporation of 5-methylycytosines and 5-hydroxymethlycytosines in iMs could be useful for fine-tuning the pH- or temperature-dependent folding/unfolding of an iM. Variations in the degree of hydration of iMs may also provide an additional control of the folded/unfolded state of iMs without having to change the pH of the surrounding matrix.

Background and purpose: Carboxylesterases (CEs) metabolize a wide range of xenobiotic substrates including heroin, cocaine, meperidine and the anticancer agent CPT-11. In this study, we have purified to homogeneity human liver and intestinal CEs and compared their ability with hydrolyse heroin, cocaine and CPT-11. Experimental approach: The hydrolysis of heroin and cocaine by recombinant human CEs was evaluated and the kinetic parameters determined. In addition, microsomal samples prepared from these tissues were subjected to chromatographic separation, and substrate hydrolysis and amounts of different CEs were determined. Key results: In contrast to previous reports, cocaine was not hydrolysed by the human liver CE, hCE1 (CES1), either as highly active recombinant protein or as CEs isolated from human liver or intestinal extracts. These results correlated well with computer-assisted molecular modelling studies that suggested that hydrolysis of cocaine by hCE1 (CES1), would be unlikely to occur. However, cocaine, heroin and CPT-11 were all substrates for the intestinal CE, hiCE (CES2), as determined using both the recombinant protein and the tissue fractions. Again, these data were in agreement with the modelling results. Conclusions and implications: These results indicate that the human liver CE is unlikely to play a role in the metabolism of cocaine and that hydrolysis of this substrate by this class of enzymes is via the human intestinal protein hiCE (CES2). In addition, because no enzyme inhibition is observed at high cocaine concentrations, potentially this route of hydrolysis is important in individuals who overdose on this agent.

Structures formed by single-strand DNA have become increasingly interesting because of their roles in a number of biological processes, particularly transcription and its regulation. Of particular importance is the fact that antitumor drugs such as Actinomycin D can selectively bind DNA hairpins over fully paired, double-strand DNA. A new fluorescent base analog, pyrrolo-deoxycytidine (PdC), can now be routinely incorporated into single-strand DNA. The fluorescence of PdC is particularly useful for studying the formation of single-strand DNA in regions of double-strand DNA. The fluorescence is quenched when PdC is paired with a complementary guanine residue, and thus is greatly enhanced upon formation of single-strand DNA. Hence, any process that results in melting or opening of DNA strands produces an increase in the fluorescence intensity of this base analog. In this study we measured the structural effects of incorporating PdC into DNA hairpins, and the effect of this incorporation on the binding of the hairpins by a fluorescent analog of the drug Actinomycin D. Two hairpin DNAs were used: one with PdC in the stem (basepaired) and one with PdC in the loop (unpaired). The thermal stability, 7-aminoactinomycin D binding, and three-dimensional structures of PdC incorporated into these DNA hairpins were all quite similar as compared to the hairpins containing an unmodified dC residue. Fluorescence lifetime measurements indicate that two lifetimes are present in PdC, and that the increase in fluorescence of the unpaired PdC residue compared to the basepaired PdC is due to an increase in the contribution of the longer lifetime to the average fluorescence lifetime. Our data indicate that PdC can be used effectively to differentiate paired and unpaired bases in DNA hairpin secondary structures, and should be similarly applicable for related structures such as cruciforms and quadruplexes. Further, our data indicate that PdC can act as a fluorescence resonance energy transfer donor for the fluorescent drug 7-aminoactinomycin D.

Carboxylesterases (CE) are ubiquitous enzymes responsible for the detoxification of xenobiotics, including numerous clinically used drugs. Therefore, the selective inhibition of these proteins may prove useful in modulating drug half-life and bioavailability. Recently, we identified 1,2-diones as potent inhibitors of CEs, although little selectivity was observed in the inhibition of either human liver CE (hCE1) or human intestinal CE (hiCE). In this paper, we have further examined the inhibitory properties of ethane-1,2-diones toward these proteins and determined that, when the carbonyl oxygen atoms are cis-coplanar, the compounds demonstrate specificity for hCE1. Conversely, when the dione oxygen atoms are not planar (or are trans-coplanar), the compounds are more potent at hiCE inhibition. These properties have been validated in over 40 1,2-diones that demonstrate inhibitory activity toward at least one of these enzymes. Statistical analysis of the results confirms the correlation (P0.001) between the dione dihedral angle and the preferential inhibition of either hiCE or hCE1. Overall, the results presented here define the parameters necessary for small molecule inhibition of human CEs.

A number of N,N′-disubstituted perylenetetracarboxylic diimides have been reported to bind effectively to DNA that adopts G-quadruplex motifs. In some cases, this binding may actively drive the transition from single-strand DNA to the quadruplex form. The perylenediimides in the reported cases all have amine-containing side chains, which are thought to interact with the grooves of the quadruplex and help dictate the selectivity of these compounds for quadruplex versus duplex DNA. We synthesized a polyethyleneglycol-swallowtailed (PEG-tailed) perylenediimide that is water-soluble even though it is uncharged. Binding to duplex and quadruplex DNA of this perylenediimide was studied by fluorescence quenching titrations under a variety of salt conditions, and the compound’s effect on quadruplex formation was studied by non-denaturing gel electrophoresis. Our results indicate that while the molecule binds to single-stranded DNA quite effectively and with selectivity, it does not drive the transition of the DNA to the tetrameric quadruplex structure, supporting the idea that charge neutralization is a key component of perylene compounds that stabilize tetrameric quadruplexes.

Carboxylesterases (CE) are ubiquitous enzymes that hydrolyze numerous ester-containing xenobiotics, including complex molecules, such as the anticancer drugs irinotecan (CPT-11) and capecitabine and the pyrethroid insecticides. Because of the role of CEs in the metabolism of many exogenous and endogenous ester-containing compounds, a number of studies have examined the inhibition of this class of enzymes. Trifluoromethylketone-containing (TFK) compounds have been identified as potent CE inhibitors. In this article, we present inhibition constants for 21 compounds, including a series of sulfanyl, sulfinyl, and sulfonyl TFKs with three mammalian CEs, as well as human acetyl- and butyrylcholinesterase. To examine the nature of the slow tight-binding inhibitor/enzyme interaction, assays were performed using either a 5-min or a 24-h preincubation period. Results showed that the length of the preincubation interval significantly affects the inhibition constants on a structurally dependent basis. The TFK-containing compounds were generally potent inhibitors of mammalian CEs, with Ki values as low as 0.3 nM observed. In most cases, thioether-containing compounds were more potent inhibitors then their sulfinyl or sulfonyl analogs. QSAR analyses demonstrated excellent observed versus predicted values correlations (r2 ranging from 0.908-0.948), with cross-correlation coefficients (q2) of approximately 0.9. In addition, pseudoreceptor models for the TKF analogs were very similar to structures and models previously obtained using benzil- or sulfonamide-based CE inhibitors. These studies indicate that more potent, selective CE inhibitors, containing long alkyl or aromatic groups attached to the thioether chemotype in TFKs, can be developed for use in in vivo enzyme inhibition.

Benzil has been identified as a potent selective inhibitor of carboxylesterases (CEs). Essential components of the molecule required for inhibitory activity include the dione moiety and the benzene rings, and substitution within the rings affords increased selectivity toward CEs from different species. Replacement of the benzene rings with heterocyclic substituents increased the K(i) values for the compounds toward three mammalian CEs when using o-nitrophenyl acetate as a substrate. Logarithmic plots of the K(i) values versus the empirical resonance energy, the heat of union of formation energy, or the aromatic stabilization energy determined from molecular orbital calculations for the ring structures yielded linear relationships that allowed prediction of the efficacy of the diones toward CE inhibition. Using these data, we predicted that 2,2'-naphthil would be an excellent inhibitor of mammalian CEs. This was demonstrated to be correct with a K(i) value of 1 nM being observed for a rabbit liver CE. In addition, molecular simulations of the movement of the ring structures around the dione dihedral indicated that the ability of the compounds to inhibit CEs was due, in part, to rotational constraints enforced by the dione moiety. Overall, these studies identify subdomains within the aromatic ethane-1,2-diones, that are responsible for CE inhibition.

Carboxylesterases (CE) are ubiquitous enzymes responsible for the metabolism of xenobiotics. Because the structural and amino acid homology among esterases of different classes, the identification of selective inhibitors of these proteins has proved problematic. Using Telik's target-related affinity profiling (TRAP) technology, we have identified a class of compounds based on benzil (1,2-diphenylethane-1,2-dione) that are potent CE inhibitors, with K(i) values in the low nanomolar range. Benzil and 30 analogues demonstrated selective inhibition of CEs, with no inhibitory activity toward human acetylcholinesterase or butyrylcholinesterase. Analysis of structurally related compounds indicated that the ethane-1,2-dione moiety was essential for enzyme inhibition and that potency was dependent on the presence of, and substitution within, the benzene ring. 3D-QSAR analyses of these benzil analogues for three different mammalian CEs demonstrated excellent correlations of observed versus predicted K(i) (r(2)0.91), with cross-validation coefficients (q(2)) of 0.9. Overall, these results suggest that selective inhibitors of CEs with potential for use in clinical applications can be designed.

The anticancer prodrug 7-ethyl-10-[4-(1-piperidino)-1-piperidino-]carbonyloxycamptothecin (CPT-11) is a highly effective camptothecin analog that has been approved for the treatment of colon cancer. It is hydrolyzed by carboxylesterases to yield 7-ethyl-10-hydroxycamptothecin (SN-38), a potent topoisomerase I poison. However, upon high-dose intravenous administration of CPT-11, a cholinergic syndrome is observed that can be ameliorated by atropine. Previous studies have indicated that CPT-11 can inhibit acetylcholinesterase (AChE), and here, we provide a detailed analysis of the inhibition of AChE by CPT-11 and by structural analogs. These studies demonstrate that the terminal dipiperidino moiety in CPT-11 plays a major role in enzyme inhibition, and this has been confirmed by X-ray crystallographic studies of a complex of the drug with Torpedo californica AChE. Our results indicate that CPT-11 binds within the active site gorge of the protein in a fashion similar to that observed with the Alzheimer drug donepezil. The 3D structure of the CPT-11/AChE complex also permits modeling of CPT-11 complexed with mammalian butyrylcholinesterase and carboxylesterase, both of which are known to hydrolyze the drug to the active metabolite. Overall, the results presented here clarify the mechanism of AChE inhibition by CPT-11 and detail the interaction of the drug with the protein. These studies may allow the design of both novel camptothecin analogs that would not inhibit AChE and new AChE inhibitors derived from the camptothecin scaffold.

Camptothecin (CPT) analogs that form more stable ternary complexes with DNA and topoisomerase I (termed cleavable complexes) show greater activity in their ability to inhibit tumor cell line growth in preclinical studies. Based on our earlier work, we hypothesized that analogs bearing hydrogen bonding moieties at the 7- through 10-position of CPT would result in more stable cleavable complexes. Consequently, we synthesized analogs with 7-mono-, 7-di-, and 7-trihydroxymethylaminomethyl groups. These analogs showed increasing cleavable complex stability as the number of hydroxyl groups was increased. The 7-trihydroxymethylaminomethyl analog of 10,11-methylenedioxycamptothecin (THMAM-MD) showed remarkable ternary complex stability with a half-life of 116 minutes. This is an order of magnitude more stable than any previously examined analog. Our in vitro analysis demonstrated that these analogs were all potent topoisomerase I poisons and could inhibit tumor cell growth in culture. We studied the effects of THMAM-MD in vivo in severe combined immunodeficient mice bearing HT-29 colon cancer and MiaPaCa-2 pancreatic cancer tumors. The THMAM-MD analog showed excellent, persisting activity in inhibiting tumor growth with both lines. Taken together, our results suggest that CPTs with hydrophilic, hydrogen-bonding groups at the 7-position hold the promise of excellent clinical activity.

Epigenetic modifications to DNA base sequences may regulate gene expression. CpG islands can contain methylated (5mC) or hydroxymethylated (5hmC) cytosine. Most CpG islands are found primarily in promoter regions that may also contain a high number of repeated cytosines and/or guanines. G-quadraplexes (G4) and i-motifs (iM) are two unique DNA secondary structures that can form in repeating sequences of either guanine or cytosine, respectively. Both G4 and iM sequences may contain CpG sequences that can be methylated or hydroxymethylated. The effects of CpG islands on DNA secondary structures were determined by incorporating a single 5hmC at varying positions in the Vascular Endothelial Growth Factor (VEGF) G4 and iM sequences. An Olis DSM-20 spectropolarimeter and a Cary 100 UV-visible spectrometer were used to monitor the effect of 5hmC on G4 and iM thermal stability. Two of the three 5hmC-containing loops showed a notable decrease in stability for G4's and increased intermolecular structure formation. Contrastingly, the iM stability increased when 5hmC was incorporated into its sequence. Additionally, there was little change in the iM pka. In summary, our results suggest the 5hmC has little effect on iM structures, but can destabilize the G4's.

Due to the diversity of biological activities that can be found in aquatic ecosystems, marine metabolites have been an active area of drug discovery for the last 30 years. Marine metabolites have been found to inhibit a number of enzymes important in the treatment of human disease. Here, we focus on marine metabolites that inhibit the enzyme acetylcholinesterase, which is the cellular target for treatment of early-stage Alzheimer’s disease. Currently, development of anticholinesterase drugs with improved potency, and drugs that act as dual acetylcholinesterase and amyloid-β aggregation inhibitors, are being sought to treat Alzheimer’s disease. Seven classes of marine metabolites are reported to possess anti-cholinesterase activity. We compared these metabolites to clinically-used acetylcholinesterase inhibitors having known mechanisms of inhibition. We performed a docking simulation and compared them to published experimental data for each metabolite to determine the most likely mechanism of inhibition for each class of marine inhibitor. Our results indicate that several marine metabolites bind to regions of the acetylcholinesterase active site that are not bound by the clinically-used drugs rivastigmine, galanthamine, donepezil, or tacrine. We use the novel poses adopted for computational drug design of tighter binding anticholinesterase drugs likely to act as inhibitors of both acetylcholinesterase activity and amyloid-β aggregation inhibition.

The antitumor antibiotic 7-aminoactinomycin D has been previously demonstrated to bind and stabilize transient hairpin structures formed by single-stranded DNA. Those experiments suggested that DNA secondary structures are viable targets for development of novel antitumor or antiviral compounds. Interestingly, when 7-aminoactinomycin D binds to hairpins formed by selected single-stranded DNAs having a high affinity for the drug, the fluorescence quantum yield, average fluorescence lifetime, excitation wavelengths, and emission wavelengths for the drug are significantly different from those observed when it is free in solution or bound to duplex, B-form DNA. This suggests a unique physical chemical environment within certain DNA hairpins that is specifically recognized by the drug. To understand the environmental contribution to the fluorescence properties of the bound drug, we used solvatochromism techniques, whereby 7-aminoactinomycin D fluorescence properties were determined in 20 solvents with known po...

Our recent work has indicated that the potent antibiotic and antitumor agent actinomycin D has the ability to selectively bind and stabilize single-stranded DNA that is capable of adopting a hairpin conformation. This mechanism of DNA binding has been implicated in the drug's ability to inhibit transcription by HIV reverse transcriptase from single-stranded DNA templates. In this report, we studied the importance of the hairpin loop on the ability of the 7-amino analog of actinomycin D to selectively bind DNA hairpins. Binding dissociation constant (Kd) values were determined to be 0.22 +/- 0.11 microM for the hairpin formed from the single-stranded DNA 5'-AAAAAAATAGTTTTAAATATTTTTTT-3' (dubbed HP1). The hairpin stem without the loop resulted in binding with Kd = 2.6 +/- 0.9 microM. The drug showed low affinity for the HP1 strand fully duplexed to its complementary sequence (estimated to be at least Kd > 21 microM). Evaluation of 7-aminoactinomycin D binding to a library of thermodynamically characterized DNA hairpins revealed an affinity for the hairpin-forming sequence 5'-GGATACCCCCGTATCC-3' (dubbed ACC4) of Kd = 6.8 +/- 2.2 microM. Replacement of the terminal guanines of this sequence to generate 5'-ATATACCCCCGTATAT-3' resulted in a 10-fold increase in affinity for this hairpin compared to ACC4, to Kd = 0.74 +/- 0.06 microM. A molecular model of the ACC4actinomycin D complex reveals that significant interactions between the hairpin loop and the pentapeptide rings of the drug must occur during drug binding. Taken together, our data indicate that the composition of the stem-loop interface is critical for the selectivity of actinomycin D and its 7-amino analog for DNA hairpins and suggests that novel drugs may be designed based on selection for the desired hairpin composition.

DNA secondary structures containing regions of single-stranded DNA have now been identified in the genomic DNA of a number of prokaryotic and eukaryotic species, including humans. Many of these secondary structures are associated with regions of DNA involved in regulation of transcription: promoters or upstream elements. The secondary structures involved appear likely to be hairpin or cruciform structures that may be recognition sites for binding of transcription factors. In the case of the coliphage N4 virion RNA polymerase, a defined hairpin in the polymerase promoter necessary for binding of the polymerase and regulation of transcription has been shown to be extruded under physiological conditions in plasmid DNA. The presence of single-stranded DNA in the promoters of several species suggests that regulatory hairpins may be involved in transcription of a number of genes. In support of this, hairpin- or cruciform-binding proteins have been identified from several species. These results imply that secondary structures in regulatory regions may be targets for drugs that bind and either block or enhance binding of proteins involved in transcription. In this review, we discuss the evidence for DNA secondary structures, particularly hairpins and cruciforms, in genomic DNA and review the studies to date of development of small molecules that can selectively bind these structures.

A planar array immunosensor, equipped with a charge-coupled device (CCD) as a detector, was used to simultaneously detect 3 toxic analytes. Wells approximately 2 mm in diameter were formed on glass slides using a photoactivated optical adhesive. Antibodies against staphylococcal enterotoxin B (SEB), ricin, and Yersinia pestis were covalently attached to the bottoms of the circular wells to form the sensing surface. Rectangular wells containing chicken immunoglobulin were used as alignment markers and to generate control signals. After removing the optical adhesive, the slides were mounted over a scientific grade CCD operating at ambient temperature in inverted (multipin phasing) mode. A two-dimensional graded index of refraction lens array was used to focus the sensing surface onto the CCD. Solutions of toxins were then placed on the slide. After rinsing, Cy5-labeled antibodies were introduced. The identity and amount of toxin bound at each location on the slide were determined by quantitative image analysis. Concentrations as low as 25 ng/mL of ricin, 15 ng/mL of pestis F1 antigen, and 5 ng/mL of SEB could be routinely measured.

G-quadruplex- and i-motif-based DNA oligomers are being investigated for their integration into therapeutic and diagnostic micro-assemblies. Examples include quadruplex-forming aptamers as potential anti-HIV agents, and serum-stable quadruplexes as carriers for delivering porphyrins into cancer cells for photodynamic therapy. The i-motifs from C-rich DNA find application in pH-triggered hydrogels that can carry agents like drugs, proteins, and polymers to their targets. The pH-dependent conformational dynamics of i-motifs also make them useful as biosensors for detecting pH changes in cellular microenvironments. Due to these and many other applications, and in an effort to present a compendia of recent uses of quadruplexes and i-motifs, this chapter will concern itself with formation of G-quadruplexes and i-motifs; their physical and chemical properties; the effects of molecular crowding and hydration on their structure and stability; and their application in therapeutics as drug targets, drug-delivery vehicles, and diagnostic tools.

The sequence specificity and structural aspects of the mode of interaction of the antitumor drug actinomycin D (AMD) with single-stranded DNA were studied by fluorescence, absorption and NMR spectroscopy, calorimetry, ultrasonic velocity and density measurements, and molecular modeling. The binding is length and sequence dependent, with the tetranucleotide motif TAGT showing the highest affinity. A “hemi-intercalation” model for the interaction is proposed.

We have studied the sequence specificity in the binding of the potent antitumor drug actinomycin D (AMD) to single-stranded DNA (ssDNA) by fluorescence and NMR spectroscopy and by molecular modeling. The significant absorption and emission changes accompanying the interaction of the fluorescent derivative 7-amino-AMD with DNAs varying in length and base composition were used to calculate affinity constants for the drug-DNA complexes. The guanine-containing trinucleotide sequences AGT, AGA, and TGT embedded within 25-base oligonucleotides, constituted favorable binding sites. In contrast, the sequence TGA did not bind the drug appreciably. Among the DNAs studied, the highest affinity was for the tetranucleotide sequence TAGT. The binding was length dependent, an oligonucleotide of at least 14 bases being required for effective complex formation (Ka > 10(4) M1=). AMD also bound to poly(d(AGT)). Gel electrophoresis confirmed that the complex was formed between the drug and individual unstructured DNA strands. The 1H NMR spectra of oligonucleotides containing the TAGT site and their complexes with AMD provided further insight into the mode(s) of interaction. A comparison of the measured chemical shifts with those estimated from ring-current calculations provided strong evidence for a hemi-intercalation of AMD between the A and G purine bases with a preference for one of two possible relative orientations. The latter were modeled as complexes with the sequence T3AGT3 and refined by force field calculations with the AMBER program. The biological implications for this novel form of interaction of AMD with single-stranded DNA are discussed.

Topological variants of single-strand DNA (ssDNA) structures, referred to as “functional DNA,” have been detected in regulatory regions of many genes and are thought to affect gene expression. Two fluorescent analogs of deoxycytidine, Pyrrolo-dC (PdC) and 1,3-diaza-2-oxophenoxazine (tC∘), can be incorporated into DNA. Here, we describe spectroscopic studies of both analogs to determine fluorescent properties that report on structural transitions from double-strand DNA (dsDNA) to ssDNA, a common pathway in the transition to functional DNA structures. We obtained fluorescence-detected circular dichroism (FDCD) spectra, steady-state fluorescence spectra, and fluorescence lifetimes of the fluorophores in DNA. Our results show that PdC is advantageous in fluorescence lifetime studies because of a distinct ~2 ns change between paired and unpaired bases. However, tC∘ is a better probe for FDCD experiments that report on the helical structure of DNA surrounding the fluorophore. Both fluorophores provide complementary data to measure DNA structural transitions.

Of the compounds that have now been shown to circumvent acquired cellular multidrug resistance, little or no structure-activity relationship has been found, although their proposed mechanism of action is through modulation of function of p-glycoprotein. While it has been suggested that this inhibition is a direct binding to p-glycoprotein, we show here that such a model seriously neglects the effects many of these compounds have on lipid physical properties. We have characterized the interactions between 16 structurally diverse pharmacological agents (nine of which are known to reverse multidrug resistance) and a variety of lipids. Potent modulators inhibit the membrane binding of rhodamine 6G, and we have observed a correlation of the measured K i values with the effectiveness of the compounds in situ. We have determined the effects of the compounds on detergent micellization, and have shown substantial changes on the critical micelle concentration of detergents in the presence of modulators. Finally, we have examined the changes in model membrane ‘viscosity’ induced by the compounds. These results indicate that both direct p-glycoprotein and indirect lipid interactions of modulators should be considered in the mechanism by which these compounds reverse multidrug resistance.

We present here the initial characterization of the mechanism of reversal of cellular resistance to Vinca alkaloids by phenoxazine (PZ). Changes in fluorescence upon cellular accumulation of PZ allowed measurement of the membrane transport kinetics in a sensitive KB-3-1 cell line and two multi-drug resistant (MDR) counterparts. The accumulation of PZ is characterized by two uptake routes, with pseudo-first order rate constants of 0.3 s−1 and 0.07 s−1, while efflux of PZ from cells revealed rate constants of 0.2 s−1. PZ rapidly reaches steady-state concentrations within cells, which may make it more clinically useful than modulators that accumulate more slowly (e.g. verapamil).

The potent RNA polymerase inhibitors actinomycin D and 7-aminoactinomycin D are shown to bind to single-stranded DNAs. The binding occurs with particular DNA sequences containing guanine residues and is characterized by hypochromic UV absorption changes similar to those observed in interactions of the drugs with double-stranded duplex DNAs. The most striking feature of the binding is the dramatic (ca. 37-fold) enhancement in fluorescence that occurs when the 7-aminoactinomycin is bound to certain single-stranded DNAs. This fluorescence of the complex is also characterized by a 40-nm hypsochromic shift in the emission spectrum of the drug and an increase in the emission anisotropy relative to the free drug or the drug bound to calf thymus DNA. The fluorescence lifetimes change in the presence of the single-stranded DNA in a manner compatible with the intensity difference. Thus, there is an increase in the fraction of the emission corresponding to a 2-ns lifetime component compared to the predominant approximately 0.5-ns lifetime of the free drug. The 7-aminoactinomycin D comigrates in polyacrylamide gels with the single-stranded DNAs, and the fluorescence of the bound drug can be visualized by excitation with 540-nm light. The binding interactions are characterized by association constants of 2.0 x 10(6) to 1.1 x 10(7) M-1.

Carboxylesterases (CE) are ubiquitous enzymes thought to be responsible for the metabolism and detoxification of xenobiotics. Numerous clinically used drugs including Demerol, lidocaine, capecitabine, and CPT-11 are hydrolyzed by these enzymes. Hence, the identification and application of selective CE inhibitors may prove useful in modulating the metabolism of esterified drugs in vivo. Having recently identified benzil (diphenylethane-1,2-dione) as a potent selective inhibitor of CEs, we sought to evaluate the inhibitory activity of related 1,2-diones toward these enzymes. Biochemical assays and kinetic studies demonstrated that isatins (indole-2,3-diones), containing hydrophobic groups attached at a variety of positions within these molecules, could act as potent, specific CE inhibitors. Interestingly, the inhibitory potency of the isatin compounds was related to their hydrophobicity, such that compounds with clogP values of1.25 were ineffective at enzyme inhibition. Conversely, analogs demonstrating clogP values5 routinely yielded Ki values in the nM range. Furthermore, excellent 3D QSAR correlates were obtained for two human CEs, hCE1 and hiCE. While the isatin analogues were generally less effective at CE inhibition than the benzils, the former may represent valid lead compounds for the development of inhibitors for use in modulating drug metabolism in vivo.

CpG islands are prone to epigenetic cytosine modification via methylation (5mC) or 5-hydroxymethylation (5hmC). Although their potential sequences and regions for occurrence in G/C-rich DNA overlap, throughout the genome 5mC and 5hmC, and 5mC and G-quadruplex (G4) and i-motif (iM) structures, are found to not be co-existent. Moreover, in some regions of DNA, 5hmCs and G4/iM forming regions have high potential to form in concurrence. However, minimal research has been done examining the effect of 5hmC incorporation into G4 and iMs in order to determine the effect of cytosine modification on non-B-DNA structures. These higher order secondary DNA formations have been described within the VEGF promoter and have great potential to silence its transcription and prevent subsequent angiogenesis. In addition, there are three cytosines in the promoter on both the guanine- and the cytosine-rich strands found in CpG sequences that have the potential for 5mhC modification. Therefore, the current study was undertaken to examine the whether there is an effect of 5hmC incorporation into G4 loops and iM stems on structure formation and stabilization. Significant changes in structure or stability in buffers with varying pH’s, cationic strength, and molecular crowding conditions were examined by circular dichroism and confirmed by UV-monitored melting. Electromobility shift assays differentiated inter- and intra-molecular structures, further identifying the number of isoforms. Notable changes in G4 dynamics were seen with two of the three loop modifications, which decreased overall stability and increased intermolecular structures. Much less marked effects were discovered for the iM structures. Ongoing studies examining the role of G4 and iM 5hmC incorporation in regulation by endogenous proteins are part of a global effort to best understand the physiological conditions regulating G4 and iM stability and function. These VEGF promoter higher order structures are promising therapeutic targets for anti-angiogenic therapy, and a comprehensive understanding of the physiological principles governing their formation will best inform future drug discovery efforts. Brooks Lab startup funds, R15, University of Mississippi School of Pharmacy Citation Format: Rhianna Morgan, Randy Wadkins, Tracy Brooks. Effects of 5-hydroxymethylcytosine epigenetic modification on G-quadruplex and i-motif structure and stability within the VEGF promoter. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1050. doi:10.1158/1538-7445.AM2015-1050

The anticancer prodrug CPT-11 is a highly effective camptothecin analog that has been approved for the treatment of colon cancer. The 2.6 angstroms resolution crystal structure of its complex with Torpedo californica acetylcholinesterase (TcAChE) demonstrates that CPT-11 binds to TcAChE and spans its gorge similarly to the Alzheimer drug, Aricept. The crystal structure clearly reveals the interactions, which contribute to the inhibitory action of CPT-11. Modeling of the complexes of CPT-11 with mammalian butyrylcholinesterase and carboxylesterase, both of which are known to hydrolyze the drug, shows how binding to either of the two enzymes yields a productive substrate-enzyme complex.

CPT-11 (irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) is an anticancer prodrug that has been approved for the treatment of colon cancer. It is a member of the camptothecin class of drugs and activation to the active metabolite SN-38, is mediated by carboxylesterases (CE). SN-38 is a potent topoisomerase I poison and is highly effective at killing human tumor cells, with IC50 values in the low nM range. However, upon high dose administration of CPT-11 to cancer patients, a cholinergic syndrome is observed, that can be rapidly ameliorated by atropine. This suggests a direct interaction of the drug or its metabolites with acetylcholinesterase (AChE). Kinetic studies indicated that CPT-11 was primarily responsible for AChE inhibition with the 4-piperidinopiperidine moiety, the major determinant in the loss of enzyme activity. Structural analogs of 4-piperidinopiperidine however, did not inhibit AChE, including a benzyl piperazine derivate of CPT-11. These results suggest that novel anticancer drugs could be synthesized that do not inhibit AChE, or alternatively, that novel AChE inhibitors could be designed based around the camptothecin scaffold.

The mechanism of cytotoxicity of the camptothecin family of antitumor drugs is thought to be the consequence of a collision between moving replication forks and camptothecin-stabilized cleavable DNA-topoisomerase I complexes. One property of camptothecin analogs relevant to their potent antitumor activity is the slow reversal of the cleavable complexes formed with these drugs. The persistence of cleavable complexes with time may be an essential property for increasing the likelihood of a collision between the replication fork and a cleavable complex, giving rise to lethal DNA lesions. In this paper, we examined a number of camptothecin analogs forming cleavable complexes with distinctly different stabilities. Absolute reaction rate analysis was carried out for each derivative. Our results indicate that the stability of the cleavable complex is dominated by the activation entropy (DeltaS++) of the reversal process. We measured the relative lipophilicity of the CPT analogs by reverse-phase HPLC, but the DeltaS++ of complex reversal is not directly related to the lipophilicity of the CPT analog being used. We suggest that solvent ordering around the 7- through 10-position of the CPT ring may be responsible for reversal rate's dependence on DeltaS++. We demonstrate that the cleavable complex stability conferred by each camptothecin analog is directly correlated with the induction of apoptosis and cytotoxicity to tumor cells.

The dose-limiting toxicity of the highly effective anticancer agent 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxy-camptothecin (irinotecan; CPT-11) is delayed diarrhea. This is thought to be caused by either bacteria-mediated hydrolysis of the glucuronide conjugate of the active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38) or direct conversion of CPT-11 to SN-38 by carboxylesterases (CE) in the small intestine. After drug administration, a very high level of CPT-11 is present in the bile; this is deposited into the duodenum, the region of the gut with the highest levels of CE activity. Hence, it is likely that direct conversion of the drug to SN-38 is partially responsible for the diarrhea associated with this agent. In an attempt to ameliorate this toxicity, we have applied Target-Related Affinity Profiling to identify novel CE inhibitors that are selective inhibitors of the human intestinal enzyme (hiCE). Seven inhibitors, all sulfonamide derivatives, demonstrated greater than 200-fold selectivity for hiCE compared with the human liver CE hCE1, and none was an inhibitor of human acetylcholinesterase or butyrylcholinesterase. Quantitative structure-activity relationship (QSAR) analysis demonstrated excellent correlations with the predicted versus experimental Ki values (r2 = 0.944) for hiCE. Additionally, design and synthesis of a tetrafluorine-substituted sulfonamide analog, which QSAR indicated would demonstrate improved inhibition of hiCE, validated the computer predictive analyses. These and other phenyl-substituted sulfonamides compounds are regarded as lead compounds for the development of effective, selective CE inhibitors for clinical applications.

We have examined the role of DNA composition in the binding of actinomycin D to single-stranded DNA. By using the fluorescent analogue 7-aminoactinomycin D, we were able to monitor binding of the drug to ssDNA with single base changes distant from the 5'-TAGT-3' site previously determined to be a high-affinity site for actinomycin D binding (Wadkins et al. (1996) J. Mol. Biol. 262, 53-68). Our binding studies indicated that secondary structures in the ssDNA were likely to be responsible for binding the drug. A series of six low-melting DNA hairpins containing all or part of the 5'-TAGT-3' binding site were synthesized. The highest Tm observed for the melting of these hairpins was 34.2 +/- 0.3 degrees C, and it depended on the length of the stem region. These metastable hairpins were stabilized by 7-aminoactinomycin D, with the drug shifting the Tm for the drug-hairpin complex to approximately 45 degrees C. The hairpins showed very high affinity (Kd approximately 0.1 microM) for 7-aminoactinomycin D, with some dependence on stem length. Digestion of the hairpins in the presence and absence of drug using mung bean nuclease, which specifically interacts with the loop region of hairpin DNA, revealed that the stable hairpins (i) contain a number of non-Watson-Crick base pairs, and (ii) undergo a conformational change in the loop region upon binding 7-aminoactinomycin D. Our results suggest that stabilization of unusual hairpins by actinomycin D may be an important aspect of the potent transcription inhibition activity of this drug.

Topographical image measures have been used to characterize the subnuclear distribution of DNA topoisomerase I in human tumor cell lines. This topographical analysis allowed a mathematical description of staining patterns to be produced that did not depend on subjective grading. The redistribution of topoisomerase I in response to increasing concentrations of topotecan was then monitored by this method. The cell lines were stained for topoisomerase I by indirect immunofluorescence methods. Digital imaging microscopy and image analysis were used to extract the nucleus from each cell, and nine parameters describing the topography of the distribution of topoisomerase I within the nucleus were computed for each. Use of multivariate analysis of variance enabled this nine-parameter set to be reduced to a single canonical variable, representing 60–90% of the observed internuclear variance. Plotting the canonical variable vs drug concentration resulted in dose–response curves that could be fitted well by a simpleEmaxmodel. From these curve fits, EC50andEmaxvalues for drug-induced redistribution of topoisomerase I were determined. Our results indicate that neither the maximum extent of topoisomerase I redistribution (Emax) nor the EC50for drug-induced redistribution correlated well with the growth inhibition produced by continuous exposure to topotecan in these cell lines. However, the EC50determined for the 1-h high-concentration exposure did reflect the growth inhibition produced in cells exposed to the drug for 1 h. The methodology described may also be generally applied to any antigen of interest.

Fiber optic biosensors have proven their ability to detect antigens rapidly in a variety of environmental and clinical samples. These biosensors are based on the technique of covalently linking antibodies to the core of an optical fiber and detecting antigen binding via measurement of fluorescence induced in the evanescent wave. One problem associated with these biosensors is the fiber-to-fiber variability in measured signal. We have addressed this problem by labeling a portion of the immobilized capture antibody with the fluorescent cyanine dye Cy5.5 (emission lambda max = 696 nm). The antigen was then labeled with fluorescent Cy5 (emission lambda max = 668 nm). Both fluorophores were excited by 635-nm light, and their emission was collected using both a fiber optic spectrometer and a biosensor optimized to collect fluorescence at two wave-lengths. The fluorescence from the Cy5.5-labeled capture antibody served as a calibration signal for each fiber and corrected for differences in optics, fiber defects, and varying amounts of capture antibody present on the fiber. Our data show that normalizing the signal measured from Cy5-labeled antigen binding to the Cy5.5 signal provides a standardization process for greatly reducing signal variance among individual fibers.

Spectroscopic methods are used to probe the interactions of several anilinoacridine analogues with calf thymus DNA over a wide range of temperatures and sodium chloride concentrations. The structurally similar compounds m-AMSA, AMSA (both active as antitumor agents), and o-AMSA (inactive as an antitumor agent) have been widely studied in their abilities to bind DNA in an intercalative manner. Recent studies from this laboratory reveal distinct differences in the thermodynamic binding mechanisms between m-AMSA and o-AMSA (Wadkins & Graves, 1989), with the m-AMSA-DNA interaction being an enthalpy-driven process while the binding of o-AMSA to DNA is characterized by more positive entropy values. To further examine the physical chemical properties associated with these compounds and their correlation with antitumor activities, an in-depth investigation into the thermodynamic parameters of these compounds and structurally related anilinoacridine analogues was performed. These studies demonstrate that substituent type and position on the aniline ring of the anilinoacridines greatly influences both the affinities of these drugs in binding to DNA and dictates whether the DNA binding is an enthalpy- or entropy-driven process. The differences in thermodynamic mechanisms of binding between the two isomers along with molecular modeling studies reveal the electronic and/or steric factors resulting from the positioning of the methoxy substituent group on the anilino ring directs the DNA-binding properties through orientation of the methanesulfonamido group at the 1' position of the aniline ring. The orientation of this substituent group may result in favorable contacts through hydrogen bonding with neighboring base pairs and ultimately influence the biological effectiveness as an antitumor agent.

The technique of photoaffinity labeling is applied to the actinomycin D system to provide a novel probe for the examination of the interactions of actinomycin D with nucleic acids. The capacity for covalent attachment of actinomycin D will aid greatly in the study of target-site specificities and the correlations of biological effects with biophysical DNA interactions. Through chemical modification of the parent actinomycin D molecule with a photoreactive azido substituent, a functional analog of the parent actinomycin D is generated having equilibrium binding properties identical to those of the parent molecule yet with the capacity to form a covalent attachment to DNA upon photolysis. The results presented here describe the noncovalent interactions of this photoreactive probe to DNA (absence of light) and compares the binding properties observed to those of the parent actinomycin D and 7-aminoactinomycin D analog. These studies demonstrate that the DNA binding properties (i.e. binding affinity, binding site size, and sequence specificity) retained by the 7-azidoactinomycin D, thus providing a suitable probe for examining actinomycin D-DNA interactions.

The equilibrium binding of the antitumor agent m-AMSA and its biologically inactive analog o-AMSA to native and synthetic DNAs are compared over a wide range of ionic strengths and temperatures. Although o-AMSA binds DNA with a higher affinity than m-AMSA it is not effective as an antitumor agent. Both m-AMSA and o-AMSA bind DNA in an intercalative manner. Indepth investigations into the thermodynamic parameters of these interactions reveal the interaction of m-AMSA with DNA to be an enthalpy driven process. In contrast, the structurally similar but biologically inactive o-AMSA binds DNA through an entropy driven process. The differences in thermodynamic mechanisms of binding between the two isomers reveal that the electronic and/or steric factors resulting from the position of the methoxy substituent group on the anilino ring directs the DNA binding properties of these compounds and ultimately the biological effectiveness as an antitumor agent.

The equilibrium binding of the antitumor agent m-AMSA (4'-(9-acridinylamino) methane-sulfon-m-ansidide) has been examined by optical methods. These studies which have focused on the low bound drug concentrations (r values less than 0.02, base pairs) reveal m-AMSA to bind calf thymus DNA in a highly cooperative manner as indicated by the initial positive slope of the Scatchard plot. In contrast, the studies on the parent 9-aminoacridine under identical conditions demonstrate that this compound binds DNA in a noncooperative (neighbor exclusion) manner. The positive cooperative binding phenomenon of m-AMSA is probed as a function of ionic concentration and shown to exist over the range of salt concentrations examined (0.01 to 0.1 M); however, the magnitude of the cooperative binding is altered. This observation of cooperativity is consistent with earlier studies on biologically active compounds and may be related to such binding parameters as binding sequence selectivity and/or structural perturbations to the DNA structure.

The four-stranded i-motif (iM) conformation of cytosine-rich DNA has importance to a wide variety of biochemical systems that range from their use in nanomaterials to potential roles in oncogene regulation. The iM structure is formed at slightly acidic pH, where hemi-protonation of cytosine results in a stable C-C+ base pair. Fundamental studies to understand iM formation from C-rich strands of DNA are described. We present a systematic characterization of the consequences of epigenetic modifications, molecular crowding, degree of hydration, and DNA sequence on the stabilities of iM-forming sequences. We used a number of biophysical techniques to characterize both the folded iM and the folding kinetics of an iM. We established a mechanism for the folding. We observed that the C-C+ hydrogen bonding of certain bases initiates the folding of the iM structure. We also observed that substitutions in the loop regions of iMs give a distinctly different kinetic signature during folding as compared to those bases that are intercalated. Our data reveal that the iM passes through a distinct intermediate form between the unfolded and folded form. In the course of determining this folding pathway, we established that the fluorescent dC analogs tC° and PdC can be used to monitor individual residues of an iM structure and can be used to determine the pKa of an iM. Our results indicate that 5-hydroxymethylation of cytosine destabilized the iMs against thermal and pH-dependent melting, while 5-methylcytosine modification stabilized the iMs. Under molecular crowding conditions, the thermal stability of iMs increased and the pKa was raised to near 7.0. Taken together, our work has laid the foundation for examining folding and structural changes in more complex iMs.