Thermalright Spitfire VGA Heatsink

Thermalright has just launched a new VGA Heatsink to its Spitfire lineup that measures 147 x 123 x 154mm and weighs just 550gm.Thermalright Spitfire VGA Heatsink comes with a unique angled design. A GPU contact base made of nickel-plated copper forms the starting point for six sintered nickel-plated heat-pipes, which then propagate perpendicular to the plane of the video-card, typically passing over other expansion cards in the system.

Thermalright Spitfire VGA Heatsink can provide silent passive cooling, or active cooling, by latching on 140 mm or 120 mm fans (fans not included in the package, fan-clips are).The package includes three types of heatsinks for the memory chips and VRM, all necessary retention mechanisms, including thumb-screws for tool-free installation. A syringe of the Chill Factor TIM is also included.

Thermalright Spitfire VGA Heatsink is compatible with most modern GPUs, including GeForce 7, 8 (excluding G80-based ones), 9 series (single GPU), Radeon HD 3800 series, HD 4800 series, and HD 5800 series. There is no support for GT200-based GeForce GTX 200 series.Its price and availability is yet to be confirm.

Salman Khan wants her to look curvy

Salman Khan signed her because his girlfriend was way too busy for his periodic film, Veer. So, Zarine Khan who looks more like a chubby Katrina Kaif, can be seen opposite Sallu in the upcoming film.

But the way she looks in the film was not her original form; the gal was slim and weighed just 54 kilos. So what happened? Salman told her to put on weight to look curvy like the women from the 18th century. And guess what she did? She indulged in the chocolate diet. In fact, Salman made sure that she ate cakes and pastries and lo and behold within three months, Zarine managed to add an extra eight kilos and the result is for everyone to see.

Reports reveal that most of the costumes that Zarine gets to flaunt in the movie are designed in a way that enhance her size and make her look more rounded and fuller.

Confirming this, a source tells a tabloid, “Veer required a leading actress who resembled a woman in the 18th century. The women of the time were full-bodied and voluptuous. Zarine was too thin for the character and hence we asked her to gain weight.” The condition that she has to gain weight was also mentioned in her contract.

Fat or slim, does she look pretty? Leave us your comments below.

Lux Sabsey Favourite Kaun Awards Show 2010

Bollywood Stars at STAR GOLD's Lux Sabsey Favourite Kaun Awards Show 2010 where bollywood stars came and ROCKED the SHOW. Bollywood Celebrities Akshay Kumar, Hrithik Roshan, Ranbir Kapoor, Priyanka Chopra, Bipasha Basu, Vivek Oberoi, Arshad Warsi, Asin, Shruti Haasan, Mugdha Godse, Tanushree Dutta, Farah Khan, Ritesh Deshmukh, Payal Rohatgi,Vidya Malvade, Adhuna Akhtar, Farhan Akhtar, Gulshan Grover, Sagarika Ghatge, Kashmira Shah, Nikita Anand, Manjari Phadnis, Ronit Roy, Divya Dutta, Rohit Roy, Tanaaz Irani, Rakesh Paul, Arjun Rampal, Prachi Desai, Shekhar Suman, Shilpa Saklani, Apoorva Agnihotri, Karishma Tanna, Ramesh Sippy, Kiran Juneja, Hard Kaur, Mansi Verma, Sanjay Gupta, Anu Gupta, Anil Sharma, Parveen Dusanj, Kabir Bedi, Ramesh Taurani, Raza Murad, Jackky Bhagnani, Vida Samadzai, Siddharth Kannan, Krishna Abhishek, Rati Pandey, Amit Sarin, Vineesha were seen at Lux Sabsey Favourite Kaun Awards Show 2010.

Marriage The First Night Artical

Sex Guide For the Bride

Even the young girls face a mental block towards sex, as during the growing up years, they are constantly told that sex is dirty and immoral. The constant badgering of a negative feeling towards sex ensures that the ritual of marriage does not necessarily make it acceptable overnight. The female mind is generally more inclined to softer and gentler aspects of romance in the man-woman relationship rather than a carnal indulgence.

The need of the hour is to be responsible enough to prepare young brides for this sensitive and intimated aspect of their lives by giving them a healthy and balanced understanding of sex and doing away with prejudices. It is imperative that they become uncomfortable with their sexuality, before tying the knot with a stranger. After all, lack of sex education and the resistance by school and college authorities can lead to half-baked knowledge. The repercussions could be pre-marital indulgence, wanton behavior, sexual abuse, pre-marital abortion, unwanted pregnancies, emotional trauma, a contraction of AIDS - the dreaded disease, and later, an unsuccessful marriage.

The loss of virginity in the Indian milieu is an issue of such sensitivity, that it can distort all perceptions and breed contempt for the girl. The price that one has to pay for sexual freedom could result in permanent damage to emotional health and well-being, vulnerability to the corrupt messages delivered by the print and electronic media alike and a confusion about the new liberated form of deviant sexuality, the goes under the guise of sexual freedom.

The buffer is the readiness of a society to impart healthy and legitimate information about sex and sexuality, an openness that fosters understanding and acceptance, a sex guide that helps the young to come face to face with their bodily needs and makes relationships meaningful.

The need for pre-marital Education

Strangely, when a marriage is arranged, the concerned families take their decision on the basis of horoscopes, religious and financial backgrounds, but perhaps never ascertain the physical and emotional compatibility of the couple which are practical and realistic grounds. A more serious approach towards marriage is required, as it is one of the most important decisions of our life. Blood group matching is of paramount significance.

For a great sex life, a healthy and clean body is a pre-requisite. Blood counts, biochemistry, semen analysis for the groom, blood groups, X-rays, sonography of the reproductive organs, specialized testing for AIDS, Hepatitis B, venereal diseases, thalassaemia, etc should be checked. Having undergone these, youngsters will definitely too able to tie the nuptial knot with more confidence and self-esteem and start their new relationship on a more sound footing.

Research shows that men and women have strong and equal sexual urges and it is the responsibility of both to fulfill each other's needs and respect each other's needs and feelings. Satisfaction and dissatisfaction of sex can contribute to the attitudes and behavior of both partners towards each other.

Sexual activity is not meant only for procreation. A gratifying sexual life imparts pleasure, enjoyment and a sense of well being. It is the most effective means of intimate communication, bonding and expression of love for each other. To create this comfortable camaraderie between partners, openness in thought, word and action is essential. Only then can feelings of pain or pleasure that the sexual activity causes can be communicated.

Ideal Sexual Relation

The ideal sexual encounter, which satisfies both man and woman, would be one in which there is adequate foreplay. Touching, feeling, whispering sweet-nothings, voicing one's preferences in an affectionate manner can make the body respond favorably. To a woman, the physical goes with the emotional. If she has anxieties, unburdening them will help her unwind. She must remember that arousal in the male is faster than that in the female. A woman's bodily rhythms differ from those of a man and it is up to her to convey readiness.

Every bride looks forward to this ultimate bonding with her loved one. As she feels pulsating excitement at the prospect of this close encounter, many a bride is intimidated at the prospect of losing her virginity. The thought of a stranger ( in most arranged marriages) fostering such intimacy which unnerves her.

The bride thus faces first marital night with mixed emotions. She looks forward to being the ideal sexual partner, reciprocating every move her husband makes. But she must realize that she too has every right to experience sexual bliss. In fact, an ideal sexual encounter is one, which satisfies both man woman and is a culmination of adequate foreplay and arousal…she must not be afraid to voice her needs and allow her to relax to the gentle touch of her lover. Only then will she be aware of the movements and positions that lead her to an orgasm.

Male and Female Sexuality - The difference?

Male sexuality is body-oriented. A man who gets sexually attracted to women need not be in love with her. It is this aspect that makes men frequent brothels or take keen interests in advertisements, movies and magazines that display the woman's anatomy. A woman's sexuality is related to her heart, the center of thought, feeling and emotion. Most women harbor sexual feelings for the man only when they love him. A handsome hunk may draw her attention but his machismo may not be enough for her to get sexually attracted to the man.

It has been said a man gives love to get sex while a woman gives sex to get love. There is no doubt that men too can be sensitive lots and love is a need for both. Quite a few men would bear a female sexuality making them emotional. Likewise, some women may carry a male sexuality. As a bride comprehends this paradox, she will understand the persistent need of a man to have sex, while she would be satisfied by a mere cuddle. A strong emotional foundation and a good amount of empathy could nip any problems in the bud.

Agony and Ecstasy of the first night

Long before the union takes place, the bride must get to know her prospective groom. Frequent meetings, dinner outings, a sharing of feelings, doubts, likes and dislikes can bring the couple closer. Unfortunately, arranged marriages often leave little room for such contact. Such marriages are merely an arrangement reached between two well-suited families, to keep up a tradition.

Armed with the legal and moral permission to have sex with his wife, a man with his body-oriented sexuality often disregards his bride's feelings and state of mind and with a false sense of bravado ends up forcing sex on her on the first night. The woman who ends up feeling awkward, uncomfortable and exploited, allows this humiliation, as orthodoxy has taught her to be a meek, submissive and tolerant. For the seeds of love to be sown, pain and conflicts have to be weeded out.

Very often, the couple is utterly exhausted at the end of an elaborate wedding ceremony. Both are strangers to each other and are often ignorant about the sexual experience. Men carry the burden of anxiety about their sexual performance. Girls have fears about the pain, the rupturing of the hymen, and the resultant bleeding, during their first sexual encounter.

There is also the fear of pregnancy. At the same time, both strongly feel that they have to live up to the first night fantasies depicted in novels and movies. With so many uncertainties and anxieties, it becomes impossible for a couple to enjoy one of the most profound experiences of their life.

If the couple is not familiar to each other, they should avoid sexual intercourse on the first night. It is the couple's own decision and postponement. The couple should postpone their first sexual intercourse, till both of them are physically and emotionaly relaxed, instead of rushing into a messy act.

They lose nothing. In fact, they may gain a great deal of mutual love and respect, which can then become the foundation of a happy married life. The secret to bliss is a deep understanding of each other's needs, leading to closeness so exciting that sex seems the most natural thing in the world. The bride can play a major role in influencing her husband's mind by keeping the communication free and frank.

She must open herself to erotic sensations, wake up all her senses and follow her innate bodily reactions and instincts and not be influenced by fantastic ideas of liberating her libido. Such an act involves the mind, body and soul and creates irresistible magnetism between the partners.

If rigidity gives away to initiative, sex will be wonderful, sensuous act and she will be left with ecstasy never felt before

Amit's Parents arranged his marriage after he passed his M.B.A. On the first night itself, his wife Ritu fled to her parents home. On asking her, she said that she could not remain married to Amit. She described him as abnormal, indecent and a pervert. This was because Ritu did not have any idea about the sexual relationship. For her, merely listening to the verbal description of the sexual act was enough to repulse Ritu. She became hysterical, every time her parents persuaded her to go back to Amit. She needed psychiatric treatment. The marriage failed. Amit got married a year later to another girl and got an understanding partner. Ritu remained single.

Need for a fresh outlook

This is a classic example of the consequences of ignorance or even incorrect knowledge about sex and the devastating effect it can have on marital life. Sex is an indelible part of every marriage. It is a natural instinct gifted to us unasked, that introduces a passionate proximity between a couple and infuses fire in their relationships. It is as normal and healthy as any other function of the body. Yet, a certain orthodoxy, feelings of self-doubt, our inadequacies, an uneasiness born of over-protection, prevent us from imparting to our daughters, the basic truths about nature's way to procreate. The bride thus remains unprepared for the big moment when she should let herself surrender to the sensations, that courses through her body and open a wonderful new world for her to explore. She reins in her emotions, when she should unleash them, controls her innate impulses when she should rejoice in her newfound sensuality and vacillates between agony and ecstasy. She is aware of the force that ignites the latent passions, knows that she can give it till it hurts but has yet to be comfortable in her own skin. She has inhibitions to shed, unanswered questions, an increasing curiosity, an inexplicable fear that wilt her and threaten her marriage.

It is time, sex was looked as a healthy expression of a powerful energy, that resides within us, which has the capacity to bring us into contact with our innermost needs and sensations, caresses and coaxes, our physical being to get in touch with our emotional self and helps us celebrate life as we have never did before.

It is only when we stop fighting against nature that we can hope for relationships to survive, work to be more fulfilling, and marriages to work. Then, there will be no room for anxiety, depression, guilt or self-condemnation. It is time we erased the discomfort, the uneasiness and the angst.

Often, parents are the ones who are responsible for breeding ignorance among their children. They are far from forthcoming and their unwillingness to speak or educate their children stems from social taboos. In Alka's case, her lack of readiness and preparation, her protected and conservative background,She being kept away form all sexual inputs consciously, sounded the knell of what could have been a fruitful nuptial bliss.

Preparing the mind

Even the young girls face a mental block towards sex, as during the growing up years, they are constantly told that sex is dirty and immoral. The constant badgering of a negative feeling towards sex ensures that the ritual of marriage does not necessarily make it acceptable overnight. The female mind is generally more inclined to softer and gentler aspects of romance in the man-woman relationship rather than a carnal indulgence.

The need of the hour is to be responsible enough to prepare young brides for this sensitive and intimated aspect of their lives by giving them a healthy and balanced understanding of sex and doing away with prejudices. It is imperative that they become uncomfortable with their sexuality, before tying the knot with a stranger. After all, lack of sex education and the resistance by school and college authorities can lead to half-baked knowledge. The repercussions could be pre-marital indulgence, wanton behavior, sexual abuse, pre-marital abortion, unwanted pregnancies, emotional trauma, a contraction of AIDS - the dreaded disease, and later, an unsuccessful marriage.

The loss of virginity in the Indian milieu is an issue of such sensitivity, that it can distort all perceptions and breed contempt for the girl. The price that one has to pay for sexual freedom could result in permanent damage to emotional health and well-being, vulnerability to the corrupt messages delivered by the print and electronic media alike and a confusion about the new liberated form of deviant sexuality, the goes under the guise of sexual freedom.

The buffer is the readiness of a society to impart healthy and legitimate information about sex and sexuality, an openness that fosters understanding and acceptance, a sex guide that helps the young to come face to face with their bodily needs and makes relationships meaningful.

The need for pre-marital Education

Strangely, when a marriage is arranged, the concerned families take their decision on the basis of horoscopes, religious and financial backgrounds, but perhaps never ascertain the physical and emotional compatibility of the couple which are practical and realistic grounds. A more serious approach towards marriage is required, as it is one of the most important decisions of our life. Blood group matching is of paramount significance.

For a great sex life, a healthy and clean body is a pre-requisite. Blood counts, biochemistry, semen analysis for the groom, blood groups, X-rays, sonography of the reproductive organs, specialized testing for AIDS, Hepatitis B, venereal diseases, thalassaemia, etc should be checked. Having undergone these, youngsters will definitely too able to tie the nuptial knot with more confidence and self-esteem and start their new relationship on a more sound footing.

Research shows that men and women have strong and equal sexual urges and it is the responsibility of both to fulfill each other's needs and respect each other's needs and feelings. Satisfaction and dissatisfaction of sex can contribute to the attitudes and behavior of both partners towards each other.

Sexual activity is not meant only for procreation. A gratifying sexual life imparts pleasure, enjoyment and a sense of well being. It is the most effective means of intimate communication, bonding and expression of love for each other. To create this comfortable camaraderie between partners, openness in thought, word and action is essential. Only then can feelings of pain or pleasure that the sexual activity causes can be communicated.

Ideal Sexual Relation

The ideal sexual encounter, which satisfies both man and woman, would be one in which there is adequate foreplay. Touching, feeling, whispering sweet-nothings, voicing one's preferences in an affectionate manner can make the body respond favorably. To a woman, the physical goes with the emotional. If she has anxieties, unburdening them will help her unwind. She must remember that arousal in the male is faster than that in the female. A woman's bodily rhythms differ from those of a man and it is up to her to convey readiness.

Every bride looks forward to this ultimate bonding with her loved one. As she feels pulsating excitement at the prospect of this close encounter, many a bride is intimidated at the prospect of losing her virginity. The thought of a stranger ( in most arranged marriages) fostering such intimacy which unnerves her.

The bride thus faces first marital night with mixed emotions. She looks forward to being the ideal sexual partner, reciprocating every move her husband makes. But she must realize that she too has every right to experience sexual bliss. In fact, an ideal sexual encounter is one, which satisfies both man woman and is a culmination of adequate foreplay and arousal…she must not be afraid to voice her needs and allow her to relax to the gentle touch of her lover. Only then will she be aware of the movements and positions that lead her to an orgasm.

Male and Female Sexuality - The difference?

Male sexuality is body-oriented. A man who gets sexually attracted to women need not be in love with her. It is this aspect that makes men frequent brothels or take keen interests in advertisements, movies and magazines that display the woman's anatomy. A woman's sexuality is related to her heart, the center of thought, feeling and emotion. Most women harbor sexual feelings for the man only when they love him. A handsome hunk may draw her attention but his machismo may not be enough for her to get sexually attracted to the man.

It has been said a man gives love to get sex while a woman gives sex to get love. There is no doubt that men too can be sensitive lots and love is a need for both. Quite a few men would bear a female sexuality making them emotional. Likewise, some women may carry a male sexuality. As a bride comprehends this paradox, she will understand the persistent need of a man to have sex, while she would be satisfied by a mere cuddle. A strong emotional foundation and a good amount of empathy could nip any problems in the bud.

Agony and Ecstasy of the first night

Long before the union takes place, the bride must get to know her prospective groom. Frequent meetings, dinner outings, a sharing of feelings, doubts, likes and dislikes can bring the couple closer. Unfortunately, arranged marriages often leave little room for such contact. Such marriages are merely an arrangement reached between two well-suited families, to keep up a tradition.

Armed with the legal and moral permission to have sex with his wife, a man with his body-oriented sexuality often disregards his bride's feelings and state of mind and with a false sense of bravado ends up forcing sex on her on the first night. The woman who ends up feeling awkward, uncomfortable and exploited, allows this humiliation, as orthodoxy has taught her to be a meek, submissive and tolerant. For the seeds of love to be sown, pain and conflicts have to be weeded out.

Very often, the couple is utterly exhausted at the end of an elaborate wedding ceremony. Both are strangers to each other and are often ignorant about the sexual experience. Men carry the burden of anxiety about their sexual performance. Girls have fears about the pain, the rupturing of the hymen, and the resultant bleeding, during their first sexual encounter.

There is also the fear of pregnancy. At the same time, both strongly feel that they have to live up to the first night fantasies depicted in novels and movies. With so many uncertainties and anxieties, it becomes impossible for a couple to enjoy one of the most profound experiences of their life.

If the couple is not familiar to each other, they should avoid sexual intercourse on the first night. It is the couple's own decision and postponement. The couple should postpone their first sexual intercourse, till both of them are physically and emotionaly relaxed, instead of rushing into a messy act.

They lose nothing. In fact, they may gain a great deal of mutual love and respect, which can then become the foundation of a happy married life. The secret to bliss is a deep understanding of each other's needs, leading to closeness so exciting that sex seems the most natural thing in the world. The bride can play a major role in influencing her husband's mind by keeping the communication free and frank.

She must open herself to erotic sensations, wake up all her senses and follow her innate bodily reactions and instincts and not be influenced by fantastic ideas of liberating her libido. Such an act involves the mind, body and soul and creates irresistible magnetism between the partners.

If rigidity gives away to initiative, sex will be wonderful, sensuous act and she will be left with ecstasy never felt before

Bollywood Hotties In SHORT DRESSES

Bollywood Hotties In SHORT DRESSES

Why Women Fake Orgasm?

Everybody heard rumours about women faking orgasms. Is that true? The majority of women have faked at least one orgasm, yet some fake almost all of them. Why do they do that? There are many reasons and the case is that there's no one to blame.

The most common are two reasons: they don't want to make their partners feel bad or they are tired and just want to end sex. Most females say that their partners are not satisfied until the girls feels orgasm, there's only one way to make them feel happy and stop the exhausting procedure - fake.

Another reason is that a typical female doesn't seek for orgasm; she desires a sexual relationship only because she wants intimacy. Still, such an attitude may make her partner feel bad. The only way out is to fake it out.

Some women never really experience orgasm while making sex, but they want their partner to feel good about himself and her. Men usually expect women to have pleasure, that's why females have no other choice. They have to fake to have a good relationship.

Loss of interest, having sex only because the partner wants to, also makes women to fake. Most females talk to their friends about such things and while they know other women act it, they do so too, because it's an easier way to have a good relationship.

When a man tries very hard, sometimes even too hard to make his woman have an orgasm, the woman usually finds it better to fake it than to disappoint her partner once again. They fear rejection if a man wouldn't understand her, they don't want to offend men because the absence of an orgasm is actually not their fault.

Some women may have health problems. If, while experiencing she becomes too sensitive and wants to stop, she fakes an orgasm in order to stop and relax. Orgasm may also cause discomfort and pain; that's a serious problem and she can't be blamed for faking. In such cases, most women prefer and enjoy orgasm more during solitary masturbation. Although they feel better while having orgasm themselves, they want to make their partner feel good too, so they act an orgasm out.

A female orgasm can be most easily faked by a good actress. There are few options: gripping something tightly; moaning loudly; breathing deeply; burying face into something (pillow); doing kegels or anything else at least remotely dramatic or climatic.

What to do if you think your partner is faking? Talk about it. Maybe she knows some special position or action that would make her happy. Don't get angry if she admits faking. Try to solve the problem, but don't get too far, don't try too hard because it may become an exhausting activity rather than an act of love.

Women act and men can do nothing about it; and maybe they shouldn't? Try to find out the real reasons of faking and discus possible ways out. Don't blame your partner for it; it's natural for women to do so from time to time. Show her your passion, love and try to understand her. ...

Best ideas for your perfect date

Probably the most superb for your unforgetable date sounds the idea about riding horses in the beach, across the water vawes in the view of sunset on the ocean...if you cant organize all those things keep the idea only about riding horses. Also could be suitable places near the forest or in some empty fields full of flowers.

Your partner will always be impressed by the oldest idea about organizing romantic dinner near the candle lights. It sounds very classical but believe me it is only up to you to make it special find out what he or she doesnt like about food and learn couple of recipes that you can make on your own. This realy wont cause you any problems because there are lots of information about cooking and serving table. If you are a guy make sure that she will come earlier and notice you cooking. It would be even more impressive than already served table.

Everyone likes going to the zoo and it would realy ensure you very romantic trip that you can always choose instead of simple going for a walk. All those animals and nature get people close together and eliminate stress. Such an interesting way of spending time together wont cost you a lot but such an evening will be memorable and pithy.

You would never delude yourself with inviting your date to a museum, ancient places of your town, castles, galeries or some cultural festivals. It even could help to know a lot about your partner, because such places oblige people to talk about peculiar things and shows his/her attitude to the world.

Finally, no matter where you go always use your imagination fly a kite, play hide and seek or paint fight, dance in the rain, draw one another or something.

by: Rugile Kiguolyte

Nucleic Acid Biochemistry Articles

Electronic Structure Of DNA Revealed For First Time

Utilizing a technique that combines low temperature measurements and theoretical calculations, Hebrew University of Jerusalem scientists and others have revealed for the first time the electronic structure of single DNA molecules.

The knowledge of the electronic properties of DNA is an important issue in many scientific areas from biochemistry to nanotechnology -- for example in the study of DNA damage by ultraviolet radiation that may cause the generation of free radicals and genetic mutations. In those cases, DNA repair occurs spontaneously via an electronic charge transfer along the DNA helix that restores the damaged molecular bonds.

In nano-bioelectronics, which is the advanced research field devoted to the study of biological molecules (to produce electrical nanocircuits, for example), it has been suggested that DNA, or its derivatives, may become used as possible conducting molecular wires in the realization of molecular computing networks which are smaller and more efficient than those produced today with silicon technology.

The knowledge that has been acquired in this project, say the researchers, may also be relevant for current attempts to develop new sophisticated, reliable, faster and cheaper ways to decode the sequence of human DNA.

In their work, the researchers were able to decode the electronic structure of DNA and to understand how the electrons distribute into the various parts of the double helix, a result that has been pursued by scientists for many years, but was previously hindered by technical problems.

Experimental and theoretical scientists worked with long and homogeneous DNA molecules at minus 195 degrees Celsius, using a scanning tunneling microscope (STM) to measure the current that passes across a molecule deposited on a gold substrate.

Then, by means of theoretical calculations based on the solution of quantum equations, the electronic structure of DNA corresponding to the measured current has been obtained. These results also suggest an identification of the parts of the double helix that contribute to the charge flow along the molecule.

The research, published in the journal Nature Materials, is a result of an international collaboration. The research was conducted by Errez Shapir and coordinated by Dr. Danny Porath at the Department of Physical Chemistry and Center for Nanoscience and Nanotechnology at the Hebrew University and by Dr. Rosa Di Felice at the S3 Center of INFM-CNR in Modena, Italy. Also collaborating in the project were Prof. Alexander Kotlyar at Tel Aviv University, who synthesized the molecules, the CINECA supercomputing center in Italy, and Prof. Gianaurelio Cuniberti at the University of Regensburg, Germany.

Hebrew University of Jerusalem. March 2008.

Nucleic Acid Biochemistry Articles

Peptide nucleic acid–DNA duplexes: Long range hole migration from an internally linked anthraquinone

Bruce Armitage, Danith Ly, Troels Koch, Henrik Frydenlund, Henrik Ørum, Hans G. Batz, and Gary B. Schuster

PNAS November 11, 1997 vol. 94 no. 23 12320-12325

Abstract

The discovery that peptide nucleic acids (PNA) mimic DNA and RNA by forming complementary duplex structures following Watson–Crick base pairing rules opens fields in biochemistry, diagnostics, and medicine for exploration. Progress requires the development of modified PNA duplexes having unique and well defined properties. We find that anthraquinone groups bound to internal positions of a PNA oligomer intercalate in the PNA–DNA hybrid. Their irradiation with near-UV light leads to electron transfer and oxidative damage at remote GG doublets on the complementary DNA strand. This behavior mimics that observed in related DNA duplexes and provides the first evidence for long range electron (hole) transport in PNA–DNA hybrid. Analysis of the mechanism for electron transport supports hole hopping.

Peptide nucleic acid (PNA) oligomers are DNA/RNA analogs (see Fig. 1) in which the natural sugar–phosphate backbone is replaced by a synthetic peptide backbone (1). PNA oligomers that contain purine and pyrimidine nucleobases hybridize with complementary DNA and RNA strands to form right-handed, double-helical complexes according to the Watson–Crick rules of hydrogen bond-mediated base pair formation (2). Although much has been learned about the structural (3) and thermodynamic (4) factors involved in hybridization, little is known about the chemical reactivity of PNA/DNA hybrids. It is crucial to understand how PNA/DNA hybrids mimic the reactions and functions of duplex DNA. Of immediate importance for their application as clinical diagnostic agents is investigation of the conductivity of DNA and its PNA analogs (5, 6).

DNA must balance the dual requirements of chemical stability and ease of transcription and replication (7). It is clear that DNA is far from inert toward a variety of different reactive species, particularly oxidizing agents. Oxidative damage to DNA produced by normal metabolism, deep-UV laser irradiation (8), gamma rays (9), or pulse radiolysis (10) accumulates at guanine residues, an effect attributed to one-dimensional migration of a radical cation (“hole”) along the DNA helix (11). Both the low oxidation potential and reactivity of the guanine radical cation contribute to the effectiveness of guanine as a trap for the migrating hole. Because guanine lesions may be the major cause of mutations (12), intense attention is focused on understanding the conductivity properties of DNA to elucidate the mechanisms by which migration of oxidative damage occurs (13). In this regard, the recent reports by Barton and coworkers are particularly important (14, 15). They describe a system consisting of a rhodium complex that is covalently linked to one end of a DNA duplex. Irradiation caused damage to the DNA more than 30 Å from where the complex was presumed to intercalate. This observation opens up exciting opportunities to study the factors that control hole migration in nucleic acids.

Photosensitizers often react with nucleic acids by single electron transfer to oxidize a base. Recent findings reveal that the light-induced reactions of a photosensitizer bound to duplex DNA by intercalation frequently generate alkali-dependent cleavage sites selectively at the 5′-G of G-purine doublets, with a strong preference for GG steps. These photosensitizers include substituted anthraquinones (16, 17), naphthalimides (18, 19), a rhodium metal complex (14), and riboflavin (20). Breslin and Schuster (17) demonstrated unambiguously that GG-selective, photoinduced damage of DNA arises by an electron transfer pathway from an intercalated anthraquinone. Time-resolved spectroscopy reveals that the excited state of the quinone accepts an electron from a base in the DNA within 20 ps of excitation (21). The base radical cation (hole) can either recombine with the electron, be trapped by reaction with water and/or oxygen, or migrate along the DNA helix to the lowest oxidation potential sites that serve as traps (22, 23).

We prepared a series of PNA oligomers with anthraquinone derivatives (AQ) covalently linked to internal positions. The ability of the quinone to photosensitize DNA damage by electron transfer when bound to the duplex by intercalation suggested it could serve a similar role in PNA-containing duplexes. The inability of common intercalators to bind to PNA (24) required that the quinone chromophore be covalently linked to the PNA backbone. The facile modification of PNA at internal residues as well as the superior hybridization properties of PNA oligomers offer distinct advantages relative to synthesis of modified DNA oligomers. Irradiation of the anthraquinone in the hybrid duplex leads to long distance hole migration and damage at GG sites in the DNA strand. Additional experiments reveal the mechanism for hole migration by its directional preference in the stacked base pairs of a PNA–DNA hybrid duplex.

Radiolabeling of DNA.

DNA oligomers, including those with 8-OxoG and abasic modifications, were purchased from Midland Certified Reagent (Midland, Texas) and were used as received. DNA oligomers were labeled at the 5′ terminus using [γ-³²P]-ATP and T4 polynucleotide kinase, according to standard procedures. The end-labeled DNA was purified by electrophoresis through a 20% denaturing polyacrylamide gel. The DNA band was excised from the gel, eluted overnight, and ethanol-precipitated.

Photocleavage Experiments.

In a typical experiment, PNA–DNA hybrids were formed by mixing together in a microcentrifuge tube PNA and unlabeled DNA (5 μM each) with 40,000-cpm labeled DNA in 200 μl of sodium phosphate buffer (10 mM phosphate, pH 7.0). The mixture was heated to 90°C for 2 min, then allowed to cool to room temperature over a period of 1 h. Two 10-μl aliquots were removed from the sample and kept in the dark, while the remainder was irradiated in a Rayonet Photoreactor (Southern New England Ultraviolet Company, Bransford, CT) equipped with eight lamps (λ = 350 nm). The sample tube was suspended from a rotating platform, and cooling air was supplied by a fan in the bottom of the unit. At the desired times, 2 × 10-μl aliquots were removed and kept in the dark. After irradiation, the DNA was precipitated with ethanol and dried. One tube from each set of aliquots was then incubated in 1 M piperidine at 90°C for 30 min. After evaporation of the piperidine and drying, all samples were suspended in denaturing loading buffer, then loaded onto a 20% denaturing polyacrylamide gel. Autoradiography was used to detect cleavage products.

Quantum Yield Determination.

A 40-μM sample of the PNA-2/DNA-3 hybrid duplex (T_m = 51°C) was prepared from stoichiometric amounts of each strand in 10 mM sodium phosphate buffer by heating the solution to 90°C for 5 min and then cooling it slowly (5 h) to room temperature. The sample was irradiated at 350 nm for 1 h in a calibrated Rayonet reactor equipped with eight 15-W lamps (light flux = 2.6 × 10⁻⁸ Eins/min⋅cm²). The samples were treated with piperidine (90°C for 30 min) and 5′-dephosphorylated with bacterial alkaline phosphatase. The 5′-AAT-3′ and 5′-GAAT-3′ fragments resulting from cleavage at the GG step of the DNA strand were separated and quantified by HPLC on a Rainin Microsorb-MV C₁₈ column (Rainin Instruments) with an acetonitrile/water/ammonium acetate mobile phase. The fragments were identified by comparison with authentic samples independently prepared.

The Structure of PNA Conjugates.

PNA oligomers were synthesized from N-Boc protected (2-aminoethyl)glycine monomers (25). The AQ-containing monomers are shown in Fig. 2. In AQ1, the anthraquinone group is linked to the peptide backbone by a single amide bond whereas for AQ2 there is an intervening glycine group between the quinone and backbone. A third monomer (Ac, used in control experiments) has an acetyl group bound to the backbone nitrogen atom of the (2-aminoethyl)glycine. The synthetic details and characterization of these compounds and the oligomers described below are reported elsewhere (unpublished work).

The PNA–AQ monomers were incorporated into 19-base oligomers (Fig. 3). The quinones are at the central position of PNA-1, equidistant from two CC sequences (sites B and C), with a third CC sequence (site A) two bases beyond site B. Hybridization of the PNA-1 with its complementary DNA-1Z oligomers gives duplexes with three GG sites to act as traps of a migrating radical cation. Placement of the quinone at the center of the duplex permits study of both the distance and directional dependence of hole migration.

A model of a PNA–DNA duplex containing an intercalated, covalently linked AQ1 group was built in sybyl 6.0 (Tripos Associates, St. Louis) using the coordinates for a PNA–DNA structure determined using NMR spectroscopy by Erikkson and Nielsen (27). One of the internal base pairs of the duplex was removed by excision of the PNA and DNA bases. The AQ carboxamide was then linked to the PNA backbone in place of the nucleobase while the DNA base was replaced by a hydrogen atom to create an abasic site. Even though this structure was not subjected to energy minimization, it clearly showed that there is space within the helix to accommodate the intercalated AQ group. This result and other experiments including thermal denaturation and phosphorescence quenching are consistent with an intercalated conformation of the AQ and are reported in detail elsewhere (unpublished work).

Light Causes Long Range DNA Damage.

The irradiation of AQs intercalated in duplex DNA gives efficient, piperidine-requiring strand cleavage selectively at GG sites by an electron transfer mechanism (16, 17). The base sequence in PNA-1/DNA-1X was specifically designed to probe for the corresponding reaction in PNA/DNA hybrids and to examine its mechanism. DNA-1X was labeled at its 5′ terminus with ³²P by standard methods (28) and hybridized with the complementary PNA oligomers. Irradiation of the PNA-1(AQ1)/DNA-1X hybrid at 350 nm lead to piperidine-requiring DNA strand cleavage at the three GG sites (Fig. 4, lane 8). Cleavage at the 3′-G is favored for site A whereas the 5′-G is favored at site B (Fig. 3); cleavage occurs with equal efficiency at each G of site C. DNA strand cleavage also is observed at the abasic residue directly opposite the AQ. The pattern of cleavage bands is identical for the PNA-1(AQ2) hybrid (Fig. 4, lane 12) although the efficiency is lower. Control experiments with PNA-1(Ac), which lacks the quinone group, show essentially no cleavage of the DNA strand at any site (Fig. 4, lanes 1–4).

Additional control experiments show that cleavage of the DNA strand results from an intramolecular reaction initiated by excitation of the quinone in the hybrid duplex. In the photocleavage experiment described above, the PNA and labeled DNA were hybridized in the presence of a stoichiometric amount of unlabeled DNA-1X (relative to PNA-1). This ensures that all of the PNA will be hybridized during the experiment, with some fraction of the hybrids containing labeled DNA-1X strands. Hybridization of PNA-1(AQ1) with labeled DNA-1X and with a 10-fold excess of unlabeled DNA-1X strongly inhibits cleavage of the labeled DNA because the fraction of PNA-1(AQ1) that is hybridized to labeled DNA-1X is 10-fold lower. However, hybridization in the presence of a stoichiometric amount of unlabeled complementary DNA and a 9-fold excess of noncomplementary single-stranded DNA has no effect on the cleavage of the labeled DNA. (The presence of a stoichiometric amount of complementary, unlabeled DNA in the latter case ensures that the PNA is hybridized while maintaining the concentration of unhybridized DNA.) The lack of inhibition in this experiment demonstrates that cleavage is not mediated by a freely diffusing intermediate because the excess of single-stranded, unlabeled DNA should effectively inhibit such a process. Finally, cleavage is still observed when excess unlabeled DNA-1X is added after hybridization with labeled DNA, demonstrating the kinetic stability of the PNA(AQ)–DNA hybrids studied in these experiments.

Selective cleavage of DNA at guanine has been observed in reactions initiated by 1-electron oxidation and by reaction of singlet oxygen (¹O₂) (29). The G-selective cleavage of the PNA–DNA hybrid cannot be caused by freely diffusing ¹O₂, but a recent report raises the possibility of one-dimensional intramolecular diffusion of ¹O₂ in a groove of duplex DNA (30), and this could account for the long range cleavage that is observed. However, this path is unlikely in the present case because the lifetime of the requisite AQ triplet is shortened by electron transfer quenching. Additional control experiments were performed that compare the cleavage of PNA-1(AQ1)–DNA hybrids caused by irradiation of methylene blue, a known ¹O₂ generator (31), with direct irradiation of the quinone in the hybrid duplex. Inspection of Fig. 5 shows cleavage of DNA upon irradiation of methylene blue that is enhanced significantly when D₂O is substituted for H₂O (compare Fig. 5, lanes 4 and 2), proving that the cleavage in this case is caused by reaction with ¹O₂ generated by excited methylene blue (32). No effect of D₂O is seen for the quinone-initiated cleavage (data not shown). It is important to note that the cleavage pattern due to the reaction of ¹O₂ is significantly different from that seen for irradiation of the quinone. In particular, densitometric analysis indicates that the ratio of cleavage of the 5′-G to the 3′-G at site A is 1.6 for ¹O₂ and 0.7 for the AQ-initiated reaction. Clearly, the long range G-selective cleavage reaction of the PNA–DNA hybrid duplex cannot involve ¹O₂ but must occur by electron transfer to generate a base radical cation and long distance migration of the hole as has been previously proposed for anthraquinones in duplex DNA.

Mechanistic interpretation of reactions with extraordinarily low quantum yields is risky because minor structural isomers or impurities can confound the analysis. The quantum yields for cleavage at the GG step (Φ_GG) by photonucleases range from 1.4% for an intercalated AQ derivative (with a 10-fold preference for cleavage at the 5′-G) (16) to 0.000005% reported for a Rh(phi)₂DMB⁺³ complex linked covalently to a 5′ terminus of duplex DNA and presumed to be intercalated (14). We determined Φ_GG in a PNA–DNA hybrid duplex by an HPLC technique.

The PNA-2(AQ1)/DNA-3 hybrid duplex (Fig. 3) was designed for measurement of Φ_GG. Cleavage at the 5′-G gives, after 5′-dephosphorylation, 5′-GAAT-3′, and cleavage at the 3′-G gives 5′-AAT-3′. These oligonucleotides are separated easily and quantified by reversed phase HPLC. In air-saturated solution, the Φ_GG for PNA-2(AQ1)/DNA-3 is 0.17%. The ratio of 5′ to 3′ G cleavage is 1:3, a preference opposite to that seen in duplex DNA. Clearly, the efficiency of radical cation generation, migration, and conversion to a piperidine-cleavable lesion is relatively high in the quinone-containing hybrid PNA–DNA duplex.

The 3′-G at site A of the PNA-1(AQ1)/DNA-1X hybrid is more than 25 Å from the AQ group, and the short linkage between the quinone and the PNA backbone prohibits direct contact between the donor (G) and acceptor (AQ), yet cleavage is observed at this and other remote sites within the duplex. One model for long range oxidative damage involves instantaneous delocalization of the radical cation, i.e., the electron is transferred directly from the GG site to the excited state photosensitizer. Selective GG cleavage in such a process would reflect a higher radical cation density at these most easily oxidized sites. An alternative mechanism postulates oxidation of a base at a distinct position (e.g., adjacent to the photosensitizer) followed by migration of the radical cation by sequential electron transfers, i.e., hole-hopping. In this model, the hole will be distributed among the various low oxidation potential sites only if the rate of hopping is faster than the rate of irreversible chemical reaction (e.g., addition of water or O₂ to the radical cation) at a particular site. The data presented thus far cannot distinguish between these two mechanistic models.

A Deep Trap Reveals the Mechanism.

Introduction of a low oxidation potential trap in the hybrid duplex allows a clear test of the mechanism for migration of oxidative damage. In particular, the central position of the AQ acceptor between two GG reaction sites (as opposed to the linkage of the photosensitizer to the duplex terminus) allows the placement of a trap so that instantaneous delocalization of the hole into the trap can be distinguished from its arrival at the trap by a series of hops. The delocalization model predicts inhibition of cleavage at all GG sites by the trap because the duplex is considered to be one continuous orbital system. Instantaneous connection of the hole and the trap decreases the likelihood of reaction at all other sites. On the other hand, migration by the hopping mechanism will exhibit a distinct directional preference for cleavage inhibition because the hole cannot “know” of the trap’s existence until it hops into it.

The ideal trap is a modified base that does not distort the structure of the PNA–DNA duplex and that has an oxidation potential significantly below that of the GG sequence. We selected the guanine derivative 7,8-dihydro-8-oxoguanine (8-OxoG; Fig. 3) as the trap on this basis. This modified base is often detected as a byproduct of oxidative damage (14, 20, 23, 31, 33–36). Although structural information is not available for PNA-containing duplexes, Williams and coworkers (37) recently determined the structure of a DNA duplex having an 8-OxoG substitution by x-ray crystallography and found that the oxidized base caused little perturbation. We found that substitution of an 8-OxoG for G on DNA in a PNA/DNA hybrid lowers the melting temperature of the duplex only 1°C. Foote and Sheu (36) report that the oxidation potential of 8-OxoG is 0.4–0.5 V below that of guanosine (as their t-butyldimethylsilyl-protected nucleoside derivatives). Considering that the difference in oxidation potential between G and A is only ≈0.1 V (38), 8-OxoG should provide a deep trap for holes in the PNA/DNA hybrid.

Photoinduced cleavage of the PNA-1(AQ1)/DNA-1T hybrid yields a cleavage pattern nearly identical to that observed for DNA-1X (Fig. 6, lane 4; compare with Fig. 4, lane 8). DNA-2 is analogous to DNA-1T except that 8-OxoG is substituted for G at site B. Irradiation of the PNA-1(AQ1)/DNA-2 hybrid shows significantly enhanced cleavage at site B, but the more dramatic effect is observed at site A, where cleavage is almost completely inhibited (lane 10). The 8-OxoG acts as a barrier to cleavage at this distal GG site. It is important to note that 8-OxoG substitution has no effect on cleavage at site C, which is located in the opposite direction from the trap. Clearly, the hole does not sense the presence of the 8-OxoG trap when the selection of migration direction occurs. Consequently, the hole cannot be in electronic contact with the trap as is required by the instantaneous delocalization model. These findings provide evidence that radical cations in a PNA–DNA duplex migrate by a discrete hopping mechanism.