Module 3: Nucleic acid structure

DNA

right hand antiparallel double helix

difference between RNA and DNA

RNA → OH on 3’ and 2’

DNA → OH on 3’

nucleotides

deoxyribose sugar, 5’ phosphate, nitrogenous base

deoxyribose

5 carbon sugar

C1’ = N-glycosidic linkage with base

C2’ = H

3’ 5’ = phosphodiester linkage joining sugars

4’ = part of backbone, not part of link

phosphodiester linkage

enzyme mediated condensation reaction → forms covalent bond between C3’ OH and C5’ of another

interstrand repulsion

negative charge on phosphate backbone causes repulsion

prevented by H-Bonds

purines

larger, 2 heterogenous rings

adenine and guanine

pyrimidines

smaller, 1 heterogenous ring

Cytosine, Thymine, Uracil

how are bases attached to sugar

nitrogen atom covalently binds to C1’

number of H-bonds in AT pairs

2

number of bonds in GC pairs

3

nucleoside

base + sugar

nucleoside nomenclature

adenosine, guanosine

cytidine, thymidine, uridine

nomenclature

deoxy/ribose + nucleoside + # of phosphates

primary structure

linear sequence of nucleotides in a NA strand

secondary structure

base paired NA

intermolecular base pairing

base pairing between 2 separate strands

intramolecular base pairing

base pairing within same strand

denaturation

alteration of native secondary, tertiary or quaternary structure of protein or NA

breaking of weak bonds

helicase

breaks H-bonds between bases converting dsDNA to ssDNA for replication

what affects Tm

%GC, sequence length, ionic strength

Tm

temp where ½ of NA is ssDNA

Td

temp where dsDNA completely separates

ionic strength

influences interstrand repulsion + Tm/d

higher Ionic strength

reduces interstrand repulsion = more stable

degradation

loss of primary structure → covalent bonds break

loss of phosphodiester link, base to C1’ glycosidic link, base ring structure

causes for degredation

thermal, chemical, enzymatic, physical

chemical degradation

if solution is to acidic/alkaline; deamination or heterocyclic ring opening

NA it acidic environments

promotes depurination → hydrolysis of glycosidic bind between sugar and purine base

NA and alkaline enviornment

hydrolysis of phosphodiester linkage (H+ given up)

enzymatic degredation

nucleases and glycosylases

nucleases

hydrolyzes phosphodiester linkage

glycosylases

hydrolyze glycosidic linkage

shearing

physical degradation → long strands = more susceptible

freeze thawing, rough handling (pipetting, vortex mixing, isolation process)

minimizing NA degradation

lyophilize DNA, buffer sol in pH 7-8, store NA at -20 or -70, aliquot NA samples to small volumes, nuclease free, gentle