L.AcknowledgmentsThe authors would like to thank Takeshi Oda, Mitsuo Takizawa, Naomi Iizawa, and Etsuko Kato for their assistance in the maintenance of our mouse populations. We would like to thank Editage for providing editorial assistance.Author ContributionsConceived and designed the experiments: MT TY TM SN. Performed the experiments: MT NT MH SN. Analyzed the data: MT NT. Contributed reagents/materials/analysis tools: MH KO. Wrote the paper: MT SN.
The emphasis on studying the interaction of methylxanthines such as theophylline, theobromine and caffeine (Fig. 1) with nucleic acids is mainly because of a) its dietary consumption b) their use as therapeutic agents. Interestingly these xanthine derivatives have interactions with steroid-receptor complex, DNA, RNA, adenosine receptor, protein kinases, and neurological behavior [1?6] which are reckoned to be pivotal for their ability to modulate the biochemical reactions by interacting with the nucleic acids or through cell signaling molecules. While probing the spectroscopic analysis of methylxanthines interaction with nucleic acids, it has been understood that caffeine known to interact with 59-adenosine monophosphate and poly riboadenylate by a parallel arrangement outside-stacked selfassociation to DNA bases [2,3], 
and report from Nafisi et.al, indicate that caffeine and theophylline bind to DNA in aqueous solution [17]. However a comparative analysis of caffeine, theophylline with the other structurally related compounds liketheobromine has 1662274 not yet been shown to understand their variance in binding efficacy with DNA, as all of them are having vital cellular activities. Moreover, the current study deals the binding interaction of all these three methylxanthines with DNA in the presence of divalent metal ions and during helix-coil transition state holding some key rationales are explicitly explored in detail. As far as the importance of theobromine is concerned it has been shown that theobromine JW-74 enhanced the antitumor activity of adriamycin with reduced toxicity [18,19]. It has also been reported that caffeine and theobromine inhibited the doxorubicin efflux from tumor cells and increased the tumoricidal activity with reduced side effect [8]. From our earlier reports it could be understood that since xanthine derivatives can interact with DNA, they can reduce the DNA-directed toxicity of certain intercalating dyes such as ethidium bromide, acridine orange and antitumor agents like cisplatin, novantrone, actinomycin D etc [16]. Moreover, co-administration of methylxanthines in cancer therapy used for the enhancement of anti-tumor agent’s activity and serving as candidates for radiosensitization are promising baselineMethylxanthines Binding with DNAFigure 1. The chemical structure of naturally occurring methylxanthines. doi:10.1371/journal.pone.0050019.gfor developing methylxanthines as potential secondary enhancers for future clinical trial [20?3]. It is worthwhile to mention here that caffeine and theophylline decrease the replication of the virus HIV-1 strain [24]. We have also demonstrated that methylxanthines can modulate the self-splicing activity of group I intron, showing both theophylline and theobromine relatively reduce the splicing activity of group I intron as compared to that of the control self-splicing reaction. However, caffeine, with structural difference of a single methyl group at the N-7 position, was not effective, and thus forming the SC66 baseline for the development.L.AcknowledgmentsThe authors would like to thank Takeshi Oda, Mitsuo Takizawa, Naomi Iizawa, and Etsuko Kato for their assistance in the maintenance of our mouse populations. We would like to thank Editage for providing editorial assistance.Author ContributionsConceived and designed the experiments: MT TY TM SN. Performed the experiments: MT NT MH SN. Analyzed the data: MT NT. Contributed reagents/materials/analysis tools: MH KO. Wrote the paper: MT SN.
The emphasis on studying the interaction of methylxanthines such as theophylline, theobromine and caffeine (Fig. 1) with nucleic acids is mainly because of a) its dietary consumption b) their use as therapeutic agents. Interestingly these xanthine derivatives have interactions with steroid-receptor complex, DNA, RNA, adenosine receptor, protein kinases, and neurological behavior [1?6] which are reckoned to be pivotal for their ability to modulate the biochemical reactions by interacting with the nucleic acids or through cell signaling molecules. While probing the spectroscopic analysis of methylxanthines interaction with nucleic acids, it has been understood that caffeine known to interact with 59-adenosine monophosphate and poly riboadenylate by a parallel arrangement outside-stacked selfassociation to DNA bases [2,3], and report from Nafisi et.al, indicate that caffeine and theophylline bind to DNA in aqueous solution [17]. However a comparative analysis of caffeine, theophylline with the other structurally related compounds liketheobromine has 1662274 not yet been shown to understand their variance in 
binding efficacy with DNA, as all of them are having vital cellular activities. Moreover, the current study deals the binding interaction of all these three methylxanthines with DNA in the presence of divalent metal ions and during helix-coil transition state holding some key rationales are explicitly explored in detail. As far as the importance of theobromine is concerned it has been shown that theobromine enhanced the antitumor activity of adriamycin with reduced toxicity [18,19]. It has also been reported that caffeine and theobromine inhibited the doxorubicin efflux from tumor cells and increased the tumoricidal activity with reduced side effect [8]. From our earlier reports it could be understood that since xanthine derivatives can interact with DNA, they can reduce the DNA-directed toxicity of certain intercalating dyes such as ethidium bromide, acridine orange and antitumor agents like cisplatin, novantrone, actinomycin D etc [16]. Moreover, co-administration of methylxanthines in cancer therapy used for the enhancement of anti-tumor agent’s activity and serving as candidates for radiosensitization are promising baselineMethylxanthines Binding with DNAFigure 1. The chemical structure of naturally occurring methylxanthines. doi:10.1371/journal.pone.0050019.gfor developing methylxanthines as potential secondary enhancers for future clinical trial [20?3]. It is worthwhile to mention here that caffeine and theophylline decrease the replication of the virus HIV-1 strain [24]. We have also demonstrated that methylxanthines can modulate the self-splicing activity of group I intron, showing both theophylline and theobromine relatively reduce the splicing activity of group I intron as compared to that of the control self-splicing reaction. However, caffeine, with structural difference of a single methyl group at the N-7 position, was not effective, and thus forming the baseline for the development.
