Nucleic Acid Structure and Hybridization Flashcards
covalent bonds
- strong! share e-
- can be polar or nonpolar
- polar covalent bonds (partial +/- charges) allow hydrogen bonds
- don’t change in water
noncovalent: ionic
- big difference in EN
- transfer of e-
- WEAK in water
noncovalent: hydrogen bonds
- weak
- polarity: H atom covalently attached to very EN atom (N, O, P; donors, D) has partial +ve charge
- partially +ve H can be attracted by another EN atom (acceptor, A), forming a weak H bond
- weak in water
noncovalent: Van der Waals interactions (AKA ____?)
london dispersion forces (LDF)
- between all types of molecules (polar and non-polar)
- cause is transient, unequal movement and dist. of e- = formation of temporary dipoles
- dipole of one molecule -> helps arrange another dipole of second molecule
- too close - repulsion; also decreases w/ distances
- works well if few atoms of a molecule are at same distance from few atoms of other molecule (esp complementary fit)
noncovalent: hydrophobic interactions
- polar and nonpolar regions of molecules have differing affinities (hydrophilic/hydrophobic)
- hydrophobic groups are subject to van der waals
- individually weak, but usually lots between molecules/parts of molecules
Hbonds and van der waals?
individually weak, strong together
in water: interactions/bonds strength ranking?
covalent > ionic > hydrogen bonds > van der waals
nitrogenous bases
purines (2 rings) A,G (mnemonic (GAP2)
pyrimidine (1 ring) C,T,U
nucleic acid structure
- nitrogenous bases
- ribose sugars
- phosphate group (PO4^-2)
ribose sugars, carbon diffs (RNA/DNA), monomer, polymer
- 3’ C always has hydroxyl group, where monomers are added during replication
- 2’C: DNA has H, RNA has OH
- monomer: deoxyribonucleotides, ribonucleotides
- polymer: DNA, RNA
phosphate group
PO4^-2
- why DNA always negatively charged
what does “nucleoside” include?
pentose sugar
nitrogenous base
NO phosphates
what does “nucleotide” include?
nucleoside (sugar + base) + phosphate
nucleotide options (3):
nucleoside monophosphate
nucleoside diphosphate
nucleoside triphosphate
naming nucleoside vs nucleotide (DNA vs RNA)
nucleoside:
- DNA: deoxyadenosine, deoxyguanosine, deoxycytidine, deoxythymidine, deoxynucleoside
- RNA: adenosine, guanosine, cytidine, uridine, nucleoside
nucleotide: (may contain fewer phosphates)
- DNA: deoxyadenosine 5’-triphosphate (dATP) etc, deoxynucleoside 5’-triphosphate (dNTP)
- RNA: adenosine 5’-triphosphate (ATP), etc, nucleoside 5’-triphosphate (NTP)
where does phosphodiester link DNA molecules together
3’ OH
5’ phosphate
reading direction for DNA?
5’ to 3’
nucleotide functions (4)
- informational molecules (DNA/RNA)
- high energy molecules (ATP/GTP)
- in coenzymes that act as cofactors for metabolic enzymes (CoA - AMP)
- regulatory/signaling molecules (cyclic AMP/GTP)
what is Chargaff’s rule?
purines = # pyrimidines
G=C, A=T
How and who - DNA structure discovery
X-ray diffraction
Rosalind Franklin
- irradiate crystallized DNA with x-ray and capture on film
- dark bars = phosphate backbone
- X pattern = helical structure
DNA is a ___-handed double helix
right!
DNA bases are ___nm apart? and ___ to each other
0.34nm apart, and parallel to each other
(anti-parallel chains)
major vs minor grooves?
major grooves have more space for binding proteins
One complete turn (DNA) means? how many nucleotides, what exact length?
- one minor groove to next minor groove
- every 10 nucleotides
- 3.4 nm in length (10 x 0.34nm = 3.4nm)
1 angstrom = ?
0.1 nm
Major and minor grooves characteristics
- binding sites for factors (regulatory, trans)
- each factor recognizes specific nucleotide sequence on DNA
- each nucleotide sequence “exposes” specific, unique distribution of acceptors and donors (like a code)
– minor groove patterns are more similar (AT vs TA, not AT vs GC) than major groves
forces that help form DNA double helix (5)
rigid phosphate backbone
- overall -ve
stacking interactions
- van der waals between bases
hydrophobic interactions
- highly -ve phosphate backbone “outside” vs nonpolar (hydrophobic) bases “inside
ionic interactions
- salts (+ve ions) stabilize phosphate backbone (DNA shielding)
H bonding
- complementary base pairing, not most energetically significant
forms of DNA (3), which one is normal?
B-DNA - normal DNA, right-handed helix
A-DNA - right- handed helix
Z-DNA - left-handed helix
unusual forms of DNA (2)
- slipped (tandem repeats) or cruciform (inverted repeats) can form if there are REPEATED DNA seq; internal complementarity
- slipping is ss loop
- cruciform is four-way junction
what level of structure is a cruciform?
secondary structure
triple helix DNA? AKA?
Hoogsteen base pairs
- formed when purines make up one strand and pyrimidines the other, then a THIRD strand can be added (binds to major groove of existing two strands
how is chromosomal DNA a dynamic structure?
- localized structural polymorphisms (chemically identical molecules with different structures)
– constant
– DNA seq
– local env - allows for recognition of DNA:
– gene expression
– DNA repair - B-DNA, A-DNA, Z-DNA can all exist together
two sequences that are not complementary will _____
not hybridize
if strands from native double helix (same original ds) reform a ds: _____
if strands from diff double helices (diff orig. ds) form a ds: _____
- renaturation
- hybridization
factors that denature DNA
- heat
- low ionic strength; promotes repulsion between -ve phosphate backbones (low salt)
- high pH; stripping of H+ shared between EN centers (NaOH)
- agents that influence H bonds
- agents that enhance solubility of hydrophobic substances
What are 2 good conditions for denaturation?
high heat, low salt (high stringency)
2 agents that influence H-bonds (covalent modifications)
- modify EN centers and block formation of H-bonds (formaldehyde, glyoxal)
2 agents that influence H-bonds (competition)
- have functional groups that can form H-bonds with EN centers (urea, formamide)
how to monitor DNA denaturation?
examine change in properties when strands separate:
- viscosity - rarely used (hard)
- absorbance (260 nm) - common in lab
absorption spectrophotometry
dsDNA absorption increasing = denaturation
Tm = melting temperature (temp at which 50% DNA is denatured usually used)
higher than Tm, approach complete denaturation
higher GC, higher Tm
why ssDNA increases absorption during absorption spectrophotometry?
- stacking of molecules is disturbed
- not strands coming apart, just bases unstacking
absorbance changes depending on stacking of purines and pyrimidine (ss and ds): 2 terms
- in ds, bases are stacked and absorbance is lower (HYPOCHROMIC)
- in ss, bases are unstacked and absorbance increases (HYPERCHROMIC)
___ regions separate first during denaturation
AT
Tm of DNA increases by ___deg C with every __% increase in GC content under normal conditions
0.4 deg C
1% increase
higher salt = ____ Tm
why?
higher Tm
salt stabilizes DNA
what is renaturation? aka?
recombination of two complementary ssDNA
hybridization
what does renaturation depend on? (6)
- DNA conc - complementary strands have to find each other
- salt conc - ionic conditions - mask repulsion forces of phosphate backbone
- temp - 20-25 deg C below Tm
- time (reaction time)
- size of DNA fragment
- complexity - simple seq renature faster
