An Introduction To DNA

An Introduction To desoxyribonucleic sexually transmitted diseasedesoxyribonucleic acid (deoxyribonucleic dot) is the genetic material of eve living organisms including some viruses. It is a dimer consists of deuce maroons that soak up upon each new(prenominal) and appear as a double helix that ar linked together covalently with each another(prenominal). Each strand is made up of similar repeating unit of measurements called nucleotides. Each nucleotide composed of collar different moieties,a 2-deoxyribose sugar,a phosphate crowd and a nitrogenous stem.1.1.1 2-Deoxyribose sugarThe 2-deoxyribose sugar, a major morphological component of DNA is a cyclic molecule .The sugars argon joined together by phosphate free radicals that form phosphodiester bonds amid third and fifth one C atoms of adjacent 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.5end of a DNA molecule is characterized by a free phosphate (P) class and the 3 end 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 are likewise located on the 5 carbon and 3-carbon of 2-deoxyribose sugar.These hydroxyl groups charge a DNA oligomer its designation of 5 and the 3 end(usually accent as three prime end and five prime end).1.1.2 Sugar-Phosphate backboneThe 2-deoxyribose sugar and a phosphate group forms the backbone in the DNA which are highly glacial and defines directionality of the molecule. The frigid hydrophilic back- bone is surrounded by a core of hydrophobic bases and is important for the stability and structure of DNA. The phosphate groups go by a negative energize that gives a concentra- tion of negative charge on the backbone of DNA and also makes DNA,a negatively charge51 Fundamentalsmolecule. The charge is also neutralised by DNA-binding 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 adenine (A), thymine (T), cytosine (C), and gua- nine(G). The bases come in two categories pyrimidines and purines. Larger nucleic acids adenine and thousand are members of a class of doubly ringed structures called purines while the smaller nucleic acids cytosine and thymine are members of a class of singly- ringed chemical substance structures called pyrimidines .A six-membered ring with two-nitrogen molecule formed a pyrimidine structure whereas purine is assumed by a nine-membered, ring with four- nitrogen molecule. Each unit of the ring constructing the base is calculateed to for specific identification. They are arranged in a particular order aprospicient the backbone of DNA to make a long chain of varying sequence that contains the code for proteins.The sequence specifies the exact genetic instructions required to create a particular organism with its own peculiar traits.61 Fundamentals1.1.4 Base Pairing in DNAThe nitrogenous bases are responsible to form double-strand of DNA in consequence of swooning enthalpy bonds and have specific shapes and hydrogen bond properties. The three hydrogen bonds form among thousand and cytosine and then denoted as G.C or C.G,depending on which is associated with the first strand. Similarly adenine and thymine also bond exclusively by coupling of two hydrogen bonds and then denoted as A.T or T.A. This uniting 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 molecules in pyrimidine-pyrimidine pairings are very far from each other that doesnt makes the hyd rogen bonding to be established. Purine-purine pairing are energetically unfavourable because the molecules are too close and create an electrostatic repulsion. The only possible pairings are GT and AC. Primary and secondary amine groups or hydroxyl groups are special K 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 evaluate 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 tertiary amine and the third formed between the 2 primary amine on guanine and the 2 c arbonyl on cytosine.1.1.5 DirectionalityThe directionality of DNA is vitally important to m any cellular work ates. 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 parallel 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 opposite 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 it possible to form strands of affiliated nucleotides.Molecular biologists keis ter 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 ad new wavetage of the above phenomenon to stop ligation of any unnecessary nucleic acid by removing the 5-phosphate with a phosphatase.1.2 DNA-Ligand adhereThe structure of DNA re renders a variety of sites where ligands may interact and bindwith DNA.The binding interaction between a dose and DNA often leads to a signi_-cant 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,anti bacterial,antipotozoal and antitumor.Modes of BindingBecause of the hard double-helical structure of DNA,drug molecule interact withDNA in a number of modes. A number of forces of varying strength involved in eachinteraction. Electrostatic forces with the phosphate backbone,sequence sensitive van derWaals interaction and hydrogen bonding interactions that occur between polar atom ofbases and hydrogen molecules are unified 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) External Binding (b) Intercalators (c) wrinkle binding (i)Major channel binders (ii)Minor line bindersExternal BindingThis type of binding extends due to electrostatic forces applied to the negatively chargephosphodiester group on 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 both changed.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 through electrostatic forces.Energy consumed tounstacked the nucleic acid bases which forms a gap between neighbouring base pairsinto which the intercalator can _t intimately.Because of small binding site,they have a littlesequence selectivity and many know intercalators shows limited selectivity for GC basepairs such as ethidium bromide which has a high a_nity towards GC site.Several otherdrugs such as propidium,proavin, anti-tum or drugs adriamycin and actinomycin Dintercalate with DNA.Groove BindersSmaller ligands preferentially binds to small(a) groove area whereas proteins and otherlarge molecules speci_cally _ts into the major groove region of DNA. They have crescent -shaped conformation due to strawman of two or more(prenominal) 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 useable group that forms hydrogen bonds at lower most part of DNA bases.They perfectly accommodate in the AT rich 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 take part in interaction.one of the most common example is Cis platin which is a surface known anti ca ncer drug.Minor Groove bindersThe most widely studied DNA interacting agents are minor groove binders that occursnaturally and also synthesize according to their sequence speci_c properties as they havepronounced binding a_nity towards AT rich groove.AT binding site is more ribbonyand deeper than GC so that all heteroaromatic rings such as furan,pyrole,benzene andImidazole of minor groove binders twisted and _t better into AT site by applying vander waals force.Hydrogen bonds of bound molecule attached to the AT base pairs tothe C-2 carbonyl oxygen of thymine or N-3 nitrogen of adenine.GC base pairs alsocontain same functional 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 forces 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 Berenilroentgenogram 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 match 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 printing 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 later on binding with theminor groove by 35 with respect 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 biology as a uorochrome on binding upon ATsite of minor groove binder as well as an intercalating drug.upon binding to GC richsequence without showing 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 issue of intermolecular interactions. It is a welldeveloped routine technique and plays an important role in analytical chemistry as wellas it has far-flung application in physics and life sciences. It deals with the mea-surement of the absorption of radiations in the ultraviolet and evident region of spec-trum.Spectroscopic techniques form the largest and the most important unity group oftechniques used in analytical chemistry,and provide a wide range of quantitative andqualitative information. All spectroscopic techniques depend on the emission or ab-sorption of electro magnetised radiations and used to determine the electronic structure ofatoms and molecules. In order to understand these techniques,it is necessary to havesome knowledge about properties of electromagnetic radiations and the nature of nuclea rand molecular aught. The ultraviolet region extends from 10 to cdnm.It is subdividedinto near ultraviolet region (200 to 400nm) and the far or vacuum ultraviolet region(10to 200 nm).The clear 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 filename extension with a wave motion and can travelin space and does not need a medium like air or pee 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,wavelength or wave numbers.The most familiar form of electromagnetic radiations is visible light which forms only asmall portion of full electromagnetic spectrum.Electromagnetic spectrumA plot which shows a number of absorption an chor rings with respect to energy 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 c overs 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 frequency region Nuclear magnetic resonance and electron spin resonance spectroscopic analysis.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 ene rgy changes associatedwith renewings 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 upcountry 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 molar absorptivity of metal com-plex,various enzyme essay,describe attenuation of solar or star(predicate) 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 statesthat the function of light indifferent by a cobwebby medium s independent of the inci-dent light assing through it.This shows that logarithm of the decrease in light intensityalong the light passage with respect to ponderousness 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 summationoflight 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 molar absorptivity of chromophoreand is equal to absorption of 1M of resultant role 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 vicissitudes in Nucleic AcidsAbsorption or emission of radiations in nucleic acid causes di_erent types of transmutations in UV-visible spectral regions and appear from n-pi* and pi -pi* transitions of purineand pyramidine bases.-* transitionLarge amount of energy required for the shifting 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 beingness transprent in the near UV such asmethane,heptane and cyclohexane that shows utmost 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 lone 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. *transitionMost of the organic compounds have a conjugate administration and shows -* transitionswith an intense strong absorption band occuring anywhere in the near UV region whichdepends upon the presence of heteroatoms substituents.These compounds also shows a slightly patrician 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 absorption and also color and located in the visibleregion..That is wherefore the solutions of metallic salts of titanium or copper are blue,whilepotassium permeganate yeilds violet solutions, and so on.1.4.4 Chemical shiftBathochromic shiftchange in max to lifelong wavelength(lower frequency)also change absorption,reectancetransmittance or emission spectrum of a molecule mostly due to substitution or final resulte_ect i.e change in polarity of firmness 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 increased solvation causes hysochromic shift.Mostly polar final results such as waterand alcohol have pronounce e_ect of hypsochromism due to broad hydrogen bondingbetween protons and the non-bonded electron pair during solvation.Hypoc hromic shiftreduction in the intensity of uv light without any change in wavelength called as hypochormice_ect which caused by the entry 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 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 are present in conjugations,sharp color can produce. A single functionalgroup or a collection of functional group s 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 swage 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 candid spectrum of a compound having onemain peak absorbing below 300nm possibly contains a very simple conjugated systemInstrumentation in UV-Visible SpectrophotometerUV-Visible spectrophotometer is a very simple to operate and able to perform quickqualitative as well as quantitative analysis.It is usuallay intentional around _ve funda-mentals parts i.e. a radiation source,a monochromater(wavele ngth selector),a samplecell(cuvette),detector and a signal processor (readout device) 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 dig anda scanning spectrophotometer with double commits.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 dissolved in a declaration and the other cell contains thesolvent alone.The detector measures the intensity of light transmitted through the sam-ple cell.Light sourceThe intensity of radiation coming from the light source varies over the entire UV-Visrange.More th an 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 the wavelength.QSAR and Drug design duodecimal structure-activity relationship (QSAR) (sometimes QSPR quantitatives tructure-property relationship) is the process by which chemical structure is quanti-tatively correlated with a well de_ned process, such as biologic 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 mathematical 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 geomorphological 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 shift 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 EffectSelection 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 very analysed .Most of the organic solvents successfully meet that criteria and solvents without having any conjugtion are very convenient for this pur pose.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 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 solvent 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