An Introduction To DNA

An Introduction To desoxyribonucleic acid deoxyribonucleic acid (deoxyribonucleic acid) is the genetic material of eve lifespan organisms including some viruses. It is a dimer consists of twain cosmic st peal that immerse upon severally other and progress as a reprise helix that ar linked together covalently with each other. Each strand is made up of sympathetic repeating units called stands. Each nucleotide composed of collar different moieties,a 2-deoxyribose sugar,a phosphate group and a nitrogenous fore.1.1.1 2-Deoxyribose sugarThe 2-deoxyribose sugar, a major structural comp wiznt of deoxyribonucleic acid is a cyclic mite .The sugars argon joined together by phosphate groups that nisus phosphodiester stick withs mingled with third and fifth carbon blood corpuscles of adjoining sugar rings.The 5 carbon of deoxyribose sugar is attached to the 3 carbon of the next, and make a network of 3 carbon and 5 carbon.5 balance of a DNA atom is char figure outerized by a go off phosphate (P) group and the 3 termination is characterized by a free hydroxyl (OH) group. It lacks an hydroxyl group at the 2 position as in a ribose therefore a sugar moiety is a 2-deoxyribose. Two free hydroxyl groups be too located on the 5 carbon and 3-carbon of 2-deoxyribose sugar.These hydroxyl groups give a DNA oligomer its designation of 5 and the 3 end(usually accent as three fix end and five prime end).1.1.2 Sugar-Phosphate backb whizzThe 2-deoxyribose sugar and a phosphate group forms the backbone in the DNA which ar highly polar and defines directionality of the mite. The polar hydrophilic back- bone is surrounded by a core of hydrophobic bases and is important for the st powerfulness and social structure of DNA. The phosphate groups give way a controvert even out that gives a concentra- tion of prohibit show on the backbone of DNA and as well as makes DNA,a negatively charge51 Fundamentalsmolecule. The charge is similarly neutralised by DNA- vexi ng proteins that contain the pos- itively charged amino acids lysine and arginine, which are attracted to the negatively charged phosphate backbone. See Fig. 1.1.Figure 1.1 DNA backbone1.1.3 Nucleic acid basesDNA contain four different nitrogenous bases that make monomer of one nucleotide different from other. These bases are antiophthalmic circumstanceor (A), thymine (T), cytosine (C), and gua- nine(G). The bases come in two categories pyrimidines and purines. Larger nucleic acids adenine and constant of gravitation are members of a screen of doubly ringed structures called purines patch the smaller nucleic acids cytosine and thymine are members of a class of one after another- ringed chemical structures called pyrimidines .A six-membe wild ring with two-nitrogen molecule formed a pyrimidine structure whereas purine is produced by a nine-membered, ring with four- nitrogen molecule. Each unit of the ring constructing the base is resulted to for specific identification. They are ar slogd in a particular order on the backbone of DNA to make a long range of varying sequence that contains the code for proteins.The sequence specifies the exact genetic instructions unavoidable to create a particular organism with its own unique traits.61 Fundamentals1.1.4 Base Pairing in DNAThe nitrogenous bases are responsible to form double-strand of DNA in consequence of weak atomic snatch 1 bonds and arrest specific shapes and total heat bond properties. The three heat content bonds form surrounded by guanine and cytosine and thence denoted as G.C or C.G,depending on which is associated with the first strand. Similarly adenine and thymine also bond exclusively by pairing of two enthalpy bonds and then denoted as A.T or T.A. This coupling up of nitrogen bases termed as complementarity.,A hydrogen bond donor need an equivalent hydrogen bond acceptor to form a hydrogen bond in the base across from it. Purines are only complementary with pyrimidines because molecu les in pyrimidine-pyrimidine pairings are rattling far from each other that doesnt makes the hydrogen bond to be established. Purine-purine pairing are energetically unfavourable because the molecules are too close and create an static repulsion. The only possible pairings are GT and AC. Primary and secondary amine groups or hydroxyl groups are common hydrogen bond donar while carbonyl and tertiary amines are common hydrogen bond acceptor groups. There are two hydrogen bonds between an AT base pair. One hydrogen bond lie between the 6 primary amine of adenine and the 4 carbonyl of thymine. The other hydrogen bond form between the 1 tertiary amine of adenine and the 2 secondary amine of thymine. On the other hand,GC base pair has three hydrogen bonds. One hydrogen bond lie between guanine with its 6 hydrogen bond accept carbonyl and cytosine having 4 hydrogen bond accepting primary amine. The second hydrogen bond also formed between guanine on 1 secondary amine and cytosine 3 tert iary amine and the third formed between the 2 primary amine on guanine and the 2 carbonyl on cytosine.1.1.5 DirectionalityThe directionality of DNA is vitally important to many cellular processes. since,double helices are necessarily directional(a strand running 5 to 3 pairs with strand running 3 to 5 )and processes such as DNA replication occur in only one direction. The two DNA strands in a duplex are anti agree and form a chemically stable structure. That is, one strand running from the 5-phosphate to 3-OH is paired with the other strand arranged with its 3-OH reversion the 5-phosphate of the first strand, and its 5-phosphate opposite the 3- OH of the first strand.71 Fundamentals1.1.6 3 end and 5 enDNA strand is inherently directional.The 3 prime end has a free hydroxyl (or phos- phate) on a 3 carbon and is called as the tail end. New nucleic acid molecules are formed by one end of 3-hydroxyl as it is ligated to the other end of 5-phosphate of a different nucleotide that make i t possible to form strands of connected nucleotides.Molecular biologists fundament use nucleotides that has a deficiency of 3-hydroxyl(dideoxyribonucleotides) to stop DNA replication .The 5 prime end has a free hydroxyl (or phosphate) on a 5 carbon in the sugar-ring and this end is called as the tail end . If a phosphate group bind with the 5 end, ligation of two nucleotides can form, with a phosphodiester bond from the 5-phosphate group to the 3-hydroxyl end of other nucleotide. ligation can also stop if the above process is eliminated. Molecular biologists have an advantage of the above phenomenon to stop ligation of any unessential nucleic acid by removing the 5-phosphate with a phosphatase.1.2 DNA-Ligand cover songThe structure of DNA represents a variety of sites where ligands may act and bindwith DNA.The binding interaction between a drug and DNA often leads to a signi_- angle modi_cation of the structure of the DNA and may have an important inuence ontheir physiological functions associated with several biologic e_ects including antivi-ral,antibacterial,antipotozoal and antitumor.Modes of BindingBecause of the complex double- helical structure of DNA,drug molecule interact withDNA in a come up of modes. A number of forces of varying strength involved in eachinteraction. Electrostatic forces with the phosphate backbone,sequence fond van derWaals interaction and hydrogen bonding interactions that occur between polar atom ofbases and hydrogen molecules are incorporated singly or in combination.To understandthe mechanism of interaction of each mode,it is best to discuss di_erent binding modesthat can act on DNA. (a) immaterial Binding (b) Intercalators (c) transmission channel binding (i) study groove binders (ii)Minor groove bindersExternal BindingThis type of binding results due to electrostatic forces apply to the negatively chargephosphodiester group along the backbone of DNA for cationic molecule.Ligand charge,hydrophobicity and size a_ect on electrostatic interactions.External binding may also bedue to either covalent or non-covalent interactions.This mode of binding is characteristics for major groocould potentially be sampled during simulations where the charge and shape of helical molecules are twain diversityd.IntercalatorsAn important class of molecules that binds to DNA are intercalators,which have beenextensively use as a anti- cancer drug.Intercalation occurs due to immersion of a ataromatic drug molecule between nucleic bases contributes to unwind DNA helix(67).Theinteraction between a positively charged intercalator and a negatively charged DNAcan be quite strong and form complex by electrostatic forces.Energy consumed tounstacked the nucleic acid bases which forms a gap between neighbouring base pairsinto which the intercalator can _t easily.Because of small binding site,they have a littlesequence selectivity and many cognize intercalators shows limited selectivity for GC basepairs such as ethidium br omide which has a high a_nity towards GC site.Several otherdrugs such as propidium,proavin, anti-tumor drugs adriamycin and actinomycin Dintercalate with DNA.Groove Binders smaller ligands preferentially binds to pocket-size groove persona whereas proteins and other bighearted molecules speci_cally _ts into the major groove voice of DNA. They have crescent -shaped conformation due to presence of two or more than than two aromatic rings that gives a conformational exibility to the molecule and makes it perfect to _t in the groove. They also possess some useful group that forms hydrogen bonds at lower to the highest degree part of DNA bases.They perfectly accommodate in the AT prosperous regions but some known groove binders show little preference towards GC site.Major Groove bindersPresence of number of hydrogen bonds on the DNA major groove enhance its recognition potential. Major groove speci_c compounds are alkylating and methylating agents and and N 7 position of guanine in the major groove collect part in interaction.one of the most common example is Cis platin which is a well known anti cancer drug.Minor Groove bindersThe most widely studied DNA interacting agents are kid groove binders that occurs by record and also synthesize according to their sequence speci_c properties as they havepronounced binding a_nity towards AT well-heeled groove.AT binding site is more thinnerand deeper than GC so that all heteroaromatic rings such as furan,pyrole,benzene andImidazole of nipper groove binders twisted and _t better into AT site by applying vander waals force.Hydrogen bonds of define molecule attached to the AT base pairs tothe C-2 carbonyl oxygen of thymine or N-3 nitrogen of adenine.GC base pairs alsocontain resembling useable groups but a steric block form by amino group of guanine inGC locations which causes hinderence to the formation of hydrogen bond on guanine atN-3 position and on cytosine at O-2 cabonyl position,prohibiting vad derWaal f orces andinhibit penetration of small molecules at GC sites of minor groove.AT site selectivity forpositively charged minor groove binders also enhanced due to high negative electrostaticpotential as compared to GC site. A number of experimental studies shows that minorgroove of B type of DNA duplexes more suitable for binding of small molecules mostoften with Dickerson-Drew sequence d(CGCGAATTCGCG) and also similar such asd(CGCAAATTTGCG).1.3.1 BerenilX-ray crystallography proof complex formation of berenil with dodecanucleotides,i.e.d(CGCGAATTCGCG) and d(CGCAAATTTGCG)which in turn shows its preferenceof binding with AT rich site of DNA minor groove and reside between three (AAT) orfour(AATT) base pairs. A number of research on berenil also con_rm its weak interac-tion and intercalating behavior.Hydrogen bonds are also formed between the amidiniumgroups and adenine N-3 or thymine O2 atoms on reverse strands of a double helical DNAoligonucleotide.Berenil is a curve shape drug which m atch the helical structure of DNAminor groove.1.3.2 PentamidineOne of the most clinically important drug,pentamidine is a synthetic antimicrobial com-pound also known aspentamidine (1,5-bis(4-amidinophenoxy)pentane,among all the mi-nor groove binders.It has been use as a secondary drug for treating aids related P.cariniipneumonia.Foot produce and X-ray crystallography shows its pronounced attachmentto DNA sites which has minimum four to _ve successive AT base pairs with the chargedamidinium group shows hydrogen bonding to O2 of thymine or N3 of adenine on oppo-site DNA strands. It contains two phenyl rings that are twisted after binding with theminor groove by 35 with watch over to each other by van der Waals forces.1.3.3 DAPIDAPI also called 4,6-diamidino-2-phenylindole(DAPI) is a synthetic,unfuse aromaticcompound is widely used in molecular(a) biology as a uorochrome on binding upon ATsite of minor groove binder as well as an intercalating drug.upon binding to GC richsequence w ithout video display any property of uorescence.X-ray structure of DAPI withd(CGCGAATTCGCG)exhibited that the drug span three base pairs and also give aclear picture of parallel attachment of phenyl and indole rings to the minor groove wallsof DNA. 1.4 UV-Visible SpectroscopySpectroscopy is a valuable tool in the study of intermolecular interactions. It is a welldeveloped routine proficiency and plays an important role in analytical chemistry as wellas it has widespread exertion in physics and life sciences. It deals with the mea-surement of the absorption of radiation therapys in the ultraviolet and visible region of spec-trum.Spectroscopic techniques form the largest and the most important single group oftechniques used in analytical chemistry,and tolerate a wide range of quantitative andqualitative information. All spectroscopic techniques depend on the emission or ab-sorption of electromagnetic radiations and used to determine the electronic structure ofatoms and molecules. In order to understand these techniques,it is necessary to havesome friendship about properties of electromagnetic radiations and the nature of atomicand molecular readiness. The ultraviolet region extends from 10 to 400nm.It is subdividedinto earnest ultraviolet region (200 to 400nm) and the far or vacuum ultraviolet region(10to 200 nm).The visible region extends from 400 to 800 nm.1.4.1 electromagnetic radiationsElectromagnetic radiations are produced by the oscillation of electric charge and mag-netic _eld residing on the atom and has its origins in atomic and molecular processes. Itvibrates perpendicular to the direction of propagation with a wave motion and can locomotein space and does not need a medium like air or water to travel through. There are variousforms of electromagnetic radiations e.g. visible,ultraviolet,infra-red, X-rays,microwavesand cosmic rays. They are characterised by frequencies,wave length or wave numbers.The most familiar form of electromagnetic radia tions is visible job little which forms only asmall portion of in full electromagnetic spectrum.Electromagnetic spectrumA plot which shows a number of absorption bands with respect to might versus wave-length has some properties yield various information and is broken into several regionscalled as Electromagnetic Spectrum.Di_erent regions of the electromagnetic spectrumprovide di_erent kinds of information as a result of interactions. Electromagnetic spec-trum covers a very wide range of electromagnetic radiation that starts from gammarays and ends on to radio waves. The boundaries between the regions are approximateand the molecular process associated with each region are quite di_erent.The regions inincreasing order of frequency are1/ radio set frequency region Nuclear magnetic resonance and electron spin resonancespectroscopy.The energy changes with change in direction of spin of a nucleus and elec-tron.2/ Micro wave regionrotational spectroscopy .Change in energy arise from transi-tions to higher energy associated with change in the rotational quantum number of themolecule. 3/Infra-red regionVibrational spectroscopy The energy changes associatedwith transitions between vibrational levels of molecules.4/Vis- ible and Ultraviolet regionElectronic spectroscopy The energy changes accom-pained with valence electrons of molecules.5/X-ray region inner electrons of an atom or a molecule invole in order to changeenergy of molecule.6/ X-ray region nuclear excitations necessary for an enegy change.1.4.2 Law of molecular AbsorptionBeer-Lambert lawAll spectrophotometric methods that measure concentration in terms of absorbance,includingdetection of proteins and nucleic acids,determine wedge absorptivity of metal com-plex,various enzyme essay,describe attenuation of solar or stellar radiation and di_er-ent metabolites based upon two basic rules,which combinely spoken as Beer-Lambertlaw.This law was basically originate by a French mathematician Lambert,which statesth at the function of well-fixed absorbed by a miasmic medium s supreme of the inci-dent mail assing through it.This shows that logarithm of the decrease in light intensityalong the light path with respect to thickness of medium which can be written as followlog10(I0/I) = klwhere I is incident light intensity,I is light path length,k is a medium constant which isfurther interpret by a Beer,a German Physicist in the same year states that the amountoflight absorbed is proportional to the number of molecules of the chromophore throughwhich the light passes.One can also says that constant K is directly proportional tothe chromophore concentration i.e. K=eC,e is the hero absorptivity of chromophoreand is equal to absorption of 1M of solution at a path length of 1 cm and their unit isM-1cm-1.Now,combinely Lambert-Beer law presented asA = lC,whereby,the term log10(I0/I) is re_ered as absorbance(A),l is the thickness of solutionand E is the molar absorption coe_cient.1.4.3 Electonic trans itions in Nucleic AcidsAbsorption or emission of radiations in nucleic acid causes di_erent types of transitionsin UV-visible spectral regions and appear from n-pi* and pi -pi* transitions of purineand pyramidine bases.-* transitionLarge amount of energy required for the chemiseing of an electron from a bonding molec-ular orbital to a * antibonding molecular orbital in the UV region.Unsaturated hydro-carbons shows this type of transition and being transprent in the near UV such asmethane,heptane and cyclohexane that shows upper limit absorbance below 200 nm dueto the fact that absorbance is equal to 1 for a thickness of 1 cm below 200nm. Similarly,water in the near UV(A=0.01 for 1cm ,at lambda =190nm)is transparent due to thepresence of -* and n-* transitions.n- *transitionThis type of transition usually occur in compounds having solitary pair of electrons andrequired energy lower than -* transition for the promotion of an n electron from anatom to an * molecular orbital.Moderate wavelength range for this transition is 150 to250 nm as 180nm for alcohols,near 190nm for ethers or halogen derivatives and in theregion of 220nm for amines. *transition closely of the organic compounds have a conjugate system and shows -* transitionswith an intense strong absorption band occuring anyplace in the near UV region whichdepends upon the presence of heteroatoms substituents.These compounds also shows a slightly blue and red shift with respect to its polarity.n- *transitionThese bands are called forbidden bands having a low molar absorptivity less than 100and originate from promotion of electron from a non bonding molecular orbital to ananti-bonding *orbital.This transition is more pronounced in molecules having a heteroatom with a lone pair of electron i.e.carbonyl which requires low energy and occur inthe regions from 270 to 300 nm. d-d transitionelectrons placed in incompletely _lled d orbitals of most of the inorganic salts are re-sponsible for transitions of weak ab sorption and also semblance and located in the visibleregion..That is why the solutions of aluminiferous salts of titanium or copper are blue,whilepotassium permeganate yeilds violet solutions, and so on.1.4.4 Chemical shiftBathochromic shiftchange in max to longer wavelength(lower frequency)also change absorption,reectancetransmittance or emission spectrum of a molecule broadly due to substitution or solvente_ect i.e change in polarity of solvent called as bathochromic shift or red shift.Solvente_ect is weak in less polar compounds as compared to polar one which can stabiliseexcited form,favours transition and causes a change in wavelength towards longer side. Hypsochromic shiftThe opposite e_ect of bathochromic shift also called as blue shift as max shift towardsthe blue end of spectrum.Unbonded electron pair lowers the energy of the n-orbitaland accessiond solvation causes hysochromic shift.Mostly polar solvents such as waterand alcohol have pronounce e_ect of hypsochromism d ue to broad hydrogen bondingbetween protons and the non-bonded electron pair during solvation.Hypochromic shift diminution in the intensity of uv light without any change in wavelength called as hypochormice_ect which caused by the origination of an auxochrome which distrots the chromophore.Forexample ,biphenyl shows lAMDAmax 252nm,Emax19,000,whereas 2,2-dimethylbiphenylshows Lambda max 270nm,Emax 800.Hyperchromic shiftThis e_ect leads to an increase in absorption of UV light at same wavelength due toappearance of an au that causes hyperchromic shift.For example,benzene shows B-bandat 256nm,Emax 200,whereas aniline oil shows B-band AT 280nm,Emax 1430.The increase of1230 in the value Emax of aniline compared to that of benzene is due to the hyperchromice_ect of the auxochrome NH2.1.4.5 Chromophore groupsOrganic compound mostly containing double bond is responsible to produce color andabsorption of ultraviolet or visible radiations as single bond is not enough to do thatbut if many a re present in conjugations,sharp color can produce. A single functionalgroup or a collection of functional groups also capable for absorption and they also actas a chromophore. A complex molecule can contain more than one chromophore so thee_ect of conjugation on the chromophore is to shift the maximum absorption to a longerwavelength .i.e. a bathochromic shift or red shift appear with an increase in absorptionintensity and the spectrum is strongly upset with respect to the superimposing e_ects ofrandom chromophores. The more the number of carbon atoms on which the conjugatedsystem is spreaded,the more the decrement in the di_erence between energy levels.andaccounts large bathchromic e_ect. A very simple spectrum of a compound having one master(prenominal) peak gripping below 300nm possibly contains a very simple conjugated system orchestration in UV-Visible SpectrophotometerUV-Visible spectrophotometer is a very simple to operate and able to perform quickqualitative as well as qua ntitative analysis.It is usuallay designed around _ve funda-mentals parts i.e. a radiation source,a monochromater(wavelength selector),a samplecell(cuvette),detector and a foretoken processor (readout tress) for measuring the absorp-tion of uv or visible radiations.These components are typically integrated in a uniqueframe work to make spectrometers for chemical analysis.Two types of UV-Visible spec-trophotometers are generally in usea _xed spectrophotometer with a single beam anda scanning spectrophotometer with double beams.Single beam spectrophotometers arehighly sensitive devices and obtaining a spectrum requires measuring the transmittanceof the sample and the blank at each wavelength separately.In the double beam spec-trophotometer,the light split into two parallel beams,each of which passes through acellone cell contains the sample turn in a solvent and the other cell contains thesolvent alone.The detector measures the intensity of light catching through the sam-ple cell. Light sourceThe intensity of radiation coming from the light source varies over the inherent UV-Visrange.More than one type of source can be used in UV-Vis spectrophotmeter which au-tomatically swap lamps when scanning between the UV and visible range .A deutriumlamp is used for the wavelengths in the UV range,a tungsten lamp is used for the wave-lengths in the visible range and alternatively for the entire UV-Visible region,a xenonlamp can be used.MonochromatorIts role is to spread the beam of light into its component wavelengths and a system ofslits focuses the desired wavelength on the sample cell.The most widely used dispersingdevice is a prism or a grating made p of quartz because quartz is transparent throughoutthe UV range.DetectorThe detector converts the intensity of light reaching it to an electrical signal.It is bynature a single channel device.Two types of detector are used,either a photomultipliertube or a semiconductor.For both of which the sensitivity depends upon th e wavelength.QSAR and Drug designQuantitative structure-activity relationship (QSAR) (sometimes QSPR quantitativestructure-property relationship) is the process by which chemical structure is quanti-tatively correlated with a well de_ned process, such as biological activity or chemicalreactivity.For example, biological activity can be expressed quantitatively as in the concentra-tion of a substance required to give a certain biological response. Additionally, whenphysicochemical properties or structures are expressed by numbers, one can form a math-ematical relationship, or quantitative structure-activity relationship, between the two.The numeric expression can then be used to predict the biological response of otherchemical structures.QSARs most general mathematical form is* Activity = f(physiochemical properties and/or structural properties)Quantitative structure-activity relationships (QSAR) represent an attempt to corre-late structural or property descriptors of compounds with activities. These physico-chemical descriptors, which include parameters to account for hydrophobicity, topology,electronic properties, and steric e_ects, are determined empirically or, more recently, bycomputational methods. Activities used in QSAR include chemical measurements andbiological assays. QSAR currently are being applied in many disciplines, with manypertaining to drug design and environmental risk assessment.ChromophoreOrganic compound mostly containing double bond is responsible to produce color and absorption of ultraviolet or visible radiations as single bond is not enough to do that but if many are present in conjugations,sharp color can produce. A single functional group or a collection of functional groups also capable for absorption and they also act as a chromophore. A complex molecule can contain more than one chromophore so the effect of conjugation on the chromophore is to shift the maximum absorption to a longer wavelength .i.e. a bathochromic shift or red s hift appear with an increase in absorption intensity and the spectrum is strongly upset with respect to the superimposing effects of random chromophores. The more the number of carbon atoms on which the conjugated system is spreaded,the more the decrement in the difference between energy levels.and accounts large bathchromic effect. A very simple spectrum of a compound having one main peak absorbing below 300nm possibly contains a very simple conjugated system such as diene or an enone whereas, if the spectrum is much mixed and also allocated in a visible region,then the molecule must contain chromophore having large red shift such as polyene ,polycyclic aromatic system etc.Solvent Effect extract of solvent used in UV-visible spectroscopy is very important. The prime requirement for a solvent is that it should be transparent to radiation over full UV range and also not absorb UV radiations in the region of substance whose spectrum is actually analysed .Most of the organic solvents s uccessfully meet that criteria and solvents without having any conjugtion are very convenient for this purpose.Among the solvents ,the water ,95% ethanol and hexane are most commonly used and are transparent in the full uv spectrum. Another valuable requirement for selecting a solvent is that it gives a nice spectrum of a set a absorption bands because polar solvent form hydrogen bonds with solute and the fine spectrum of the complex may vanish but this is not the case for non polar solvents where a fine spectrum often easily appears because of the absence seizure of hydrogen bonding.Polar solvents also shows bathochromic effect which causes a decrease in electronic state.Asecond criteria for agood solvent is its effect on the fine strusture of an absorption band.Ano polar solvent doesnot hydrogen bond with the solute,and the spectrum of the solute closely approximate s the spectrum that would be produced in the gaseous state ,in which fine structure is often observed.In a polar sol vent the hydrogen bonding forms a solute solvent comlex and the fine structure may disappear.Athird criteria for a good solvent is its ability to influence th