nuclic acid

1. 1
Chemistry of Nucleic Acids

2. Nucleic Acids(NAs)
- Are non-protein nitrogenous substances made up of nucleotides.
- Are non branched biopolymers.
• 2-types in living organisms: DNA & RNA.
- Units are deoxyribonucleotide for DNA & ribonucleotide for RNA.
Deoxyribonucleic Acid (DNA):
- found in every nucleated cell & carry genetic information.
- polymer of nucleotides linked by 3'→5'–phosphodiester bonds.
- found in chromosomes, mitochondria & chloroplasts.
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3. - Nuclear DNA is found bound to basic proteins(histones).
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4. Structure of DNA
- Exists as a double stranded (ds) molecule;
- except few viruses that contain single-stranded (ss) DNA.
1. In-Eukaryotes: in nucleus as nucleoproteins/chromosomes.
2. In Prokaryotes:protein–DNA complex is present in non
membrane bound region called nucleoid.
A. 3'→5'-Phosphodiester bond
- Chain has polarity,with 5'-end(free PO4) & a 3’-end(free-OH).
- bases in the chain are conventionally written from 5’-3’-end.
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5. Cont…
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- the bond is cleaved by chemicals & nucleases .
- deoxyribonucleases for DNA &
- ribonucleases for RNA.
- only RNA is cleaved by alkali.
B. Double helix:2 chains in DNA are coiled in anti-parallel manner around a
common axis called axis of symmetry.
In DNA helix: - Sugar-Po4(hydrophilic part) form back bone.
- The hydrophobic bases are stacked inside.

6. Cont…
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1) Base pairing:according to Watson & CricK
- A=T,G≡C(H-bond).
- hydrophobic interaction b/n bases.
- so, one polynucleotide chain of dsDNA helix is always the comp-
lement of the other & bps are perpendicular to axis of the helix.
 Chagraff’s Rule:in any sample of dsDNA,
- the number of A=T,& G=CPurines=Pyrimidines.

7. Cont…
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DNA double helix Base Pairing
A=T
G≡C

8. Cont…
2. Separation of the 2-DNA strands in the double helix:
- disruption of H-bonds by pH change & heat.
- When DNA is heated, the To at w/c ½ of the helical structure is lost
melting temperature (Tm).
- DNA rich in A&T has low Tm;DNA rich in G&C has high Tm.
- loss of helical structure is called DNA denaturation.
- at temp above Tm DNA exists as single stranded.
- denaturation is monitored by measuring absorbance at 260 nm.
- ss-DNA has a higher relative absorbance at 260nm than dsDNA.
- Hyperchromicity?hypochromicity? renaturation/Reannealing of DNA?
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9. Structural Forms of DNA
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There are 3 forms of DNA as described by Watson-Crick.
a) B-form: the most common form physiologically.
- planes of bases are at 90o to the axis.
- 1o component of chromosomal-DNA.
b) A-form:-Produced by moderately dehydrating the B-form.
- not found under physiological conditions.
- Found in DNA-RNA hybrids or RNA-RNA-ds regions.
c) Z-form:Longer & thinner than B-form,
- The deoxyribose–phosphate backbone forms zigzag “Z”-DNA.

10. Forms of DNA
Feature B-DNA(most
common)
A-DNA Z-DNA
- Type of helix - R.handed R.handed Left handed
- Helical Diameter (nm) 2.37 2.55 1.84
- Rise per bp(nm) 0.34 0.29 0.37
- Distance for complete
turn/pitch(nm)/360o
3.4 3.2 4.5
- Number of bp/turn 10 11 12
- Topology of Major
Groove
Wide, deep Narrow, deep Flat, convex(non
existent)
- Topology of Minor
Groove
Narrow, shallow Broad, shallow Narrow, Deep
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11. Cont…
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Base’s Helical twist Angle?

12. Cont…
- Stretches of Z-DNA can occur naturally in regions of DNA that
have a sequence of alternating purines & pyrimidines;
- For example,poly GC(GCGCGCGCGCGC…).
- NB:in A & B forms the base & the sugar are in opposite direction
to N-glycosidic linkage(anti-conformation).
- but, in the presence of high [cations] some rotates to syn form
 DNA with d/t form (the chain zigzags b/n syn at C & anti at
G-base, in Z-DNA).
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13. Cont…
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-A & B forms do have both minor & major grooves but in Z-form
deep minor groove & non existent or convex major groove.
- Grooves are sites of attachments of DNA binding proteins for
DNA replication and transcription.

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The grooves are lined by
potential H-bond donors (blue)
& acceptors (red).
Naked Extracellular
DNA(eDNA)?

15. C. Shape of DNA (Linear & Circular)
 In Eukaryotes’ nucleus 1,long & linear dsDNA bound to histone&
non-histonechromatins.
- Circular DNA:In mithocondria & chloroplasts.
 Prokaryotes:Single, ds,supercoiled,circular chromosome.
- DNA is bound to non histone proteins & condensenucleoid.
- bacteria also contain small,circular,extrachromosomal DNA
molecules called plasmids.
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16. a. 1oStructure of DNA(linear sequence deoxyribonucleotides)
- 4 d/t types of deoxyribonucleotides linked by 3’-5’.
- bases are projected laterally(sequenced 5’-3’) with 2 free ends.
b. 2o Structure of DNA:1st proposed by Watson & Crick in 1953.
- a double helix formed by the 2-strands around a central axis.
- The 2 strands coiled around the central axis.
- One strand with 5’-3’ & the other with 3’- 5’.
- one strand is coding/sense strand and the other is non-coding
/antisense strand.
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17. Cont…
- The 2-Sugar-P back bones wind around the outside of the bases & are
exposed to the aqueous solution & hydrophobic bases stacked inside.
c.3o structure of DNA when DNA wrap around histone=nucleosome.
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Secondary DNA

18. Organization of Eukaryotic DNA
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19. Histones(Globins)
- Closely related basic proteins in chromatin.
 Fun: compacting DNA(~2m)in a micro env’t(in nucleus~10μm).
- Rich in AAs like Arg& Lys(both ~25% of all AA residues).
- In Eukaryotes DNA wound around histone octamerNucleosome.
- Linker DNA(50Bp long):linking Neighboring nucleosomes.
- 5 classes of histones in eukaryotes
- d/ce is Mwt & AA sequence H1,H2A,H2B,H3,H4.
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20. Cont…
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- Nucleosomes resemble beads(Histones) on a string(DNA strand).
- Nucleosomes are further arranged into more complex structures
called chromosomes that can be segregated during cell division.
- The complex of DNA & protein=chromatin.

21. Interactions of Histones
- H3 & H4 form a tetramer containing 2 mol each=(H3H4)2
- H2A & H2B = H2A–H2B-dimer.
- Under physiological conditions,histone oligomers associatehistone
octamer=(H3H4)2(H2AH2B)2 = 8 histones/bead.
- In nucleosome,DNA is supercolied(left helix) on the octamer.
- Nucleosome ~200Bps in w/c 146 bps form 1.75 turn around the
octamer & the remaining serves as a linker DNA where H1-binds.
- Fun:formation of nucleosome protects the DNA from digestion by a
nuclease.
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22. Cont…
-
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Nuclease:endonuclease or exonuclease, -
dexy ribonuclease or ribonuclease ?
- Total length of human DNA
when summed up ~10billion
miles.
- Distance of the sun from
The earth 93million miles.

23. Amino acid Sequence in Histones
- H3 & H4 are nearly identical in AA sequence in all eukaryotes.
- H1,H2A&H2B show less sequence similarity among eukaryotes.
 H1 is not found in nucleosome core but binds with linker-DNA b/n
the nucleosome beads.
2. Higher levels of organization
- Nucleosomes packed more tightly to form a polynucleosome
(nucleofilament)often known as 30nm fiber;thenloops that are
anchored by a nuclear scaffold proteins.
- Further organization final chromosomal structure.
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24. Cont…
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25. Mitochondrial DNA:
- to synthesize Proteins of mitochondrial matrix & membrane.
- double stranded like nuclear DNA but circular.
- Mitochondrial eve? Mitochondrial genetic disease?
Satellite DNAs:very short highly repetitive sequences(exist as
tandem repeats e.g, A-T-T-C-G-A-T-T-C-G-A-T-T-)differ in
density from the remaining chromosomal DNA and form their own
band when treated with CsCl2 density gradient centrifugation.
- In man,4 satellite DNAs constitute 6% of the chromosomal DNA.
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26. Junk DNA/Non-coding DNA
- The idle portion of DNA of chromosome or genome.
- ~ 80-90% of human genome with no well understood function.
- Most sequences within introns & intergenic DNA is Junk.
- includes Transposons that encode proteins,with no clear value.
- Protein-coding DNA genes makes up barely less than 2% of the
human genome, the remaining is considered as “junk”.
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27. Role & Function of Junk DNA
- Segments of junk DNA called LINE-1 elements are capable of
repairing broken strands of DNA.
 LINE-1(long interspersed nuclear elements)~17% of human genome.
-Are transposable(change their position) elements and 80-100% are inactive.
- May be an important genetic basis for evolution.
- Antifreeze protein gene in a species of fish w/c is evolved from junk
DNA(study of Illinois University) for survival in fridge zone.
- May play a role in the regulation of gene expression & gene diversity.
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28. Ribonucleic Acid(RNA)
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- Polymer of ribonucleotide & often exists as single stranded.
- Found:Nucleolus,Nissl’s granules,ribosomes,mitochondria & cytosol.

29. Structure of RNA
a. 1oStructure:-Sequence of ribonucleotides in chain.
b. 2o Structure:
- forms Coil of the polyribonucleotide chain/hairpin loops.
- Stabilised by hydrophobic interactions of purine &pyrimidine bases.
- there are intrachain hydrogen bonds b/n G-C & A-U.
- the H-bonds are the same as in DNA for G–C while N3 as well as
C4-Oxo Group of Uracil/dihydrouracil w/c pairs with adenine.
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30. Cont…
c) 3o Structure of RNA:
- Involves the folding of the molecule into 3D-structure.
- Stabilised by hydrophobic & H-bonds producing a compactly
coiled globular structure.
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31. Types of RNAs
- 3 Major types with major function of protein synthesis.
1. Messenger RNA (m-RNA):
- the most heterogeneous class of RNA in size & stability.
- Mwt=3×104-2×106 /1000-10,000 ribonucleotides.
- formed from DNA template strand(3’–5’)during transcription.
- mRNA carries a specific sequence of nucleotides in “triplets”
called codons,responsible for specific protein synthesis.
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32. Cont…
 Poly-A tail at its 3’-end (to keep stability of mRNA by
protecting it from 3’-exonuclease attack).
 Its 5’-end is capped by 7-methylguanosine triphosphate.
- involved in recognition of protein synthesis & helps in stabilizing
mRNA by preventing the attack of 5'–exonucleases.
- The protein synthesis begins at 5’-end of the capped mRNA.
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33. hnRNA(hetergenous Nuclear RNA)
- Precurssor of cytoplasmic mRNA after cleavage by spliceosomes.
Chxs of hnRNA
- Synthesised in the nucleus, ½ life = 23 minutes.
- 10-100 X bigger than mRNA.
- bound to macromolecular proteins called informers & exists as
“heterogeneous ribonuclear proteins”(hn-RNP).
- 75% of hnRNA is degraded in the nucleus.
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34. Cont…
- Only 25% forms a precursor of mRNA(Pre-mRNA).
PRE-mRNA:-converted to mRNA.
- Has regions called introns transcripts(inactive) &
- Exons transcripts(active portion required for translation).
- ~ 80% of pre-mRNA is removed as introns & only 20% of Pre-
mRNA,the exons are spliced to form mRNA.
2) tRNA/Soluble/s-RNA:remain mainly in cytoplasm.
- relatively Small,Single-Stranded,globular molecules.
- at least 20 d/t tRNAs.
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35. Cont…
a.1o Structure of tRNA:
~75 nucleotides of A,G,C,U,pseudouridine(ψ)
& Thymine are present in one loop.
b) 2o Structure of t-RNA:- ss-tRNA molecule
folded to form a clover-leaf 2o structure.
- stabilized by H-bonds b/n complementary bases
in d/t portions of the same strand.
- these ds-helical structures are called stems.
- All tRNAs have 4 arms or loops.
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Clover leaf

36. 1.Acceptor Arm/acceptor end:-Consists of unpaired CCA at 3’-end.
- the 3’–OH terminal of Adenine may bind with the α-COOH of a specific
AA & later aminoacyl-tRNA complex to ribosomes for protein synthesis.
- formed by a base-paired acceptor stem whose bases are H-bonded with
the last few bases at the 5’ end of t-RNA.
2.Anticodon Arm:
- Unpaired/non-bonded loop carrying triplet called Anticodon.
- bases are H-bonded with 3-complementary bases of Codon of mRNA.
- The base pair-stem leading to anticodon loop called anticodon stem.
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37. Cont…
3. D arm:-b/c it contains the base dihydrouridine.
- a recognition site for aminoacyl-tRNA synthetase,w/c catalyse the
aminoacylation of the tRNA molecule.
4. TψC arm(7bases):-Contains Thymine,pseudouridine(ψ) & C.
ribosomal/r-RNA:~ 80% the total cellular RNA.
- Ribosome is a nucleoprotein found in cytoplasm.
- On ribosome the mRNA & rRNA interact during translation.
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38. Cont…
- Mammalian Ribosomes(80S) are made up of 2-subunits:
a) the larger-60S(~60% rRNA):
- combination of 5Sr-RNA,5.8S r-RNA & 28S r-RNA and 49proteins.
b) the smaller-40S(carries 18S r-RNA & 33-d/t proteins).
- the fun of rRNA in ribosomes isn’t clearly known.
- but play a role in binding of mRNA to ribosomes during translation.
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39. Regulation of synthesis of Ribosomes(r-RNA):
- Ribosomes aren’t produced unless for use by cells.
- If there are no AAs, cells stop the synthesis of ribosomes &
- If AAs are available,the control is relaxed & synthesis takes place.
 Mechanism:2-Nucleotides control
• ppGpp:-Guanosine diphosphate 2'(or 3')diphosphate
• pppGpp:-Triphosphate derivative.
- When there is no AA these nucleotides turn off r-RNA synthesis.
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40. Small RNAs
- Complexed with proteinsribonucleoproteins & distributed in nucleus
- Size 20-300 nucleotides & 100,000-1,000,000/cell).
A. snRNAs (Small nuclear RNAs):
- involved in mRNA processing & gene regulation.
- u1,u2,u4,u5 & u6 are involved in intron removal & processing of
hnRNAmRNA.
B. miRNAs(Micro RNAs):are ss-RNA molecules of (~21-23 ntds long).
- involve in gene regulation.
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41. Formation & Processing of miRNA
- Genes encoding miRNAs are much longer than the processed miRNA.
- miRNAs are 1st formed as Pre-miRNAs with a cap & a poly-A tail &
- then processed to short Pre-miRNA in nucleus by micro-processor
protein complex (Nuclease Drosha & dsRNA binding protein Pasha).
- Pre-miRNAsmiRNAs in cytosol by Endonuclease dicer.
- Initiates the formation of the RNA induced silencing complex(RISC).
- RISCGene Silencing(miRNAs hybridizeimperfect RNA-RNA
duplexes in 3’-untranslated regions of target mRNAsTranslation
arrest).
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42. Cont…
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43. C. Si-RNAs (Small Interfering RNAs)
- Play an important role in gene regulation.
- Formed by specific nucleolytic cleavage of larger dsRNAs.
- Short SiRNAs(21-23-Nucleotides)usually form perfect RNA-
RNA hybrids anywhere in mRNA where it complements.
- these duplexes of SiRNA & mRNA↓es protein production.
- SiRNA-mRNA complexes are degraded by specific nucleolytic
machinery in specific cytoplasmic organelles termed “p-bodies”.
- P-bodies=Processing bodies.
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44. Clinical Aspect
1. Therapeutic Drug Development:miRNAs &siRNAs are getting attention.
2. To ↓se/Knock Down Specific Protein Levels:siRNAs are used to ↓se/knock down”
specific protein levels via Si-RNA homology-directed mRNA degradation.
- Useful & powerful alternative to gene knockout technology.
3. miRNA & Cancer:-miRNA is carcinogen ?linked with various Ca’s as confirmed on
mice. Inhibitory effect too ?
4.miRNA & Heart Disease:Mice deficient in a muscle-specific miRNA had high rate
of developing Ventricular Septal defect.
5. Si-RNA Used for Killing Cancer Cells:Confirmed on rats.
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