Nucleic Acid Structure and Hybridization

Question 1

covalent bonds

Answer 1

• strong! share e-
• can be polar or nonpolar
• polar covalent bonds (partial +/- charges) allow hydrogen bonds
• don’t change in water

Question 2

noncovalent: ionic

Answer 2

• big difference in EN
• transfer of e-
• WEAK in water

Question 3

noncovalent: hydrogen bonds

Answer 3

• 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

Question 4

noncovalent: Van der Waals interactions (AKA ____?)

Answer 4

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)

Question 5

noncovalent: hydrophobic interactions

Answer 5

• 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

Question 6

Hbonds and van der waals?

Answer 6

individually weak, strong together

Question 7

in water: interactions/bonds strength ranking?

Answer 7

covalent > ionic > hydrogen bonds > van der waals

Question 8

nitrogenous bases

Answer 8

purines (2 rings) A,G (mnemonic (GAP2)
pyrimidine (1 ring) C,T,U

Question 9

nucleic acid structure

Answer 9

• nitrogenous bases
• ribose sugars
• phosphate group (PO4^-2)

Question 10

ribose sugars, carbon diffs (RNA/DNA), monomer, polymer

Answer 10

• 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

Question 11

phosphate group

Answer 11

PO4^-2
- why DNA always negatively charged

Question 12

what does “nucleoside” include?

Answer 12

pentose sugar
nitrogenous base
NO phosphates

Question 13

what does “nucleotide” include?

Answer 13

nucleoside (sugar + base) + phosphate

Question 14

nucleotide options (3):

Answer 14

nucleoside monophosphate
nucleoside diphosphate
nucleoside triphosphate

Question 15

naming nucleoside vs nucleotide (DNA vs RNA)

Answer 15

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)

Question 16

where does phosphodiester link DNA molecules together

Answer 16

3’ OH
5’ phosphate

Question 17

reading direction for DNA?

Answer 17

5’ to 3’

Question 18

nucleotide functions (4)

Answer 18

• 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)

Question 19

what is Chargaff’s rule?

Answer 19

purines = # pyrimidines
G=C, A=T

Question 20

How and who - DNA structure discovery

Answer 20

X-ray diffraction
Rosalind Franklin
- irradiate crystallized DNA with x-ray and capture on film
- dark bars = phosphate backbone
- X pattern = helical structure

Question 21

DNA is a ___-handed double helix

Answer 21

right!

Question 22

DNA bases are ___nm apart? and ___ to each other

Answer 22

0.34nm apart, and parallel to each other
(anti-parallel chains)

Question 23

major vs minor grooves?

Answer 23

major grooves have more space for binding proteins

Question 24

One complete turn (DNA) means? how many nucleotides, what exact length?

Answer 24

• one minor groove to next minor groove
• every 10 nucleotides
• 3.4 nm in length (10 x 0.34nm = 3.4nm)

Question 25

1 angstrom = ?

Answer 25

0.1 nm

Question 26

Major and minor grooves characteristics

Answer 26

- 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

Question 27

forces that help form DNA double helix (5)

Answer 27

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

Question 28

forms of DNA (3), which one is normal?

Answer 28

B-DNA - normal DNA, right-handed helix A-DNA - right- handed helix Z-DNA - left-handed helix

Question 29

unusual forms of DNA (2)

Answer 29

- 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

Question 30

what level of structure is a cruciform?

Answer 30

secondary structure

Question 31

triple helix DNA? AKA?

Answer 31

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

Question 32

how is chromosomal DNA a dynamic structure?

Answer 32

- 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

Question 33

two sequences that are not complementary will _____

Answer 33

not hybridize

Question 34

if strands from native double helix (same original ds) reform a ds: _____ if strands from diff double helices (diff orig. ds) form a ds: _____

Answer 34

- renaturation - hybridization

Question 35

factors that denature DNA

Answer 35

- 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

Question 36

What are 2 good conditions for denaturation?

Answer 36

high heat, low salt (high stringency)

Question 37

2 agents that influence H-bonds (covalent modifications)

Answer 37

- modify EN centers and block formation of H-bonds (formaldehyde, glyoxal)

Question 38

2 agents that influence H-bonds (competition)

Answer 38

- have functional groups that can form H-bonds with EN centers (urea, formamide)

Question 39

how to monitor DNA denaturation?

Answer 39

examine change in properties when strands separate: - viscosity - rarely used (hard) - absorbance (260 nm) - common in lab

Question 40

absorption spectrophotometry

Answer 40

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

Question 41

why ssDNA increases absorption during absorption spectrophotometry?

Answer 41

- stacking of molecules is disturbed - not strands coming apart, just bases unstacking

Question 42

absorbance changes depending on stacking of purines and pyrimidine (ss and ds): 2 terms

Answer 42

- in ds, bases are stacked and absorbance is lower (HYPOCHROMIC) - in ss, bases are unstacked and absorbance increases (HYPERCHROMIC)

Question 43

___ regions separate first during denaturation

Answer 43

AT

Question 44

Tm of DNA increases by ___deg C with every __% increase in GC content under normal conditions

Answer 44

0.4 deg C 1% increase

Question 45

higher salt = ____ Tm why?

Answer 45

higher Tm salt stabilizes DNA

Question 46

what is renaturation? aka?

Answer 46

recombination of two complementary ssDNA hybridization

Question 47

what does renaturation depend on? (6)

Answer 47

- 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