Lecture 10 – Structural Transitions in

Polynucleic Acids I
Introduction
 Structural Transitions in Nucleic Acids
– can also be modeled using statistical thermodynamics.
 We will generally adopt the Zipper Model:
– in which multiple nucleation events are unlikely…
• so that conformations contain exactly 1 helix-island.
– Model has 2 parameters:
• propagation parameter (s):
– describes the energetic favorability/residue of the transition:
s = exp[-∆Gores/RT].
• nucleation parameter (σ):
– describes the cooperativity of the transition:
» due to ‘extra’ energy required to form the ‘junction’.
• here, 0 　 < σ << 1 is the Zipper model.
– Parameter values will again differ, with transition type.
Nucleic Acid Transitions
 Nucleic Acids participate in many interesting
transitions:
– several analogous to polypeptide coil-helix and helix-coil
transitions:
• annealing of single-stranded DNAs (ssDNAs)
– to form double-stranded (ds) B-DNA helices.
– and the reverse-process (DNA melting).
• folding of ssRNA to form double-stranded A-helical regions.
– others involve transitions b/w two well-defined helices:
• the B-DNA to A-DNA transition.
• the B-DNA to Z-DNA transition.
– within linear and circular DNA.
 First, we consider dsDNA melting/annealing.
 DNA Melting and Annealing Curves
 Both the DNA melting and annealing transitions are
characterized by a midpoint temperature:
– where the fraction of single-stranded (ss) DNAs = ½.
• Tm = melting temperature.
• Ta = annealing temperature.
– these curves are often distinct:
• so that Tm > Ta.
• particularly for long DNAs…
– and fast cooling rates.
• this is called ‘hysteresis’.
 Hysteresis expresses structural
differences:
– between ½-melted DNAs and ½-annealed DNAs.
• differences in kinetic mechanism.
• as well as the degree of irreversibility in melting.
– We first consider the DNA melting process…
• modeled as a reverse transition, using the Zipper model.
DNA Melting: The Zipper Model
 Simplest case: homo-duplex melting:
– i.e., dsDNA consisting of 1 kind of base-pair.
– Similar to the polypeptide helix-coil transition,
• although the molecular details differ.
 We apply the Zipper model:
– where we neglect strand-separation.
• forward transition: coil to helix (annealing).
– melting treated as the reverse transition:
• from a fully helical state, H.
– in which all base-pairs adopt a helical state, h.
– i.e., H = …hhhhhh…
• to a fully melted state, C.
– in which all base-pairs adopt a coil state, c.
– i.e., C = …cccccc…
– Application requires definition of:
• a nucleation parameter, σ.
• a propagation parameter, s.
Ensemble Average Fraction of h’s
 DNA Melting/Annealing: Aligned Zipper Model...
– Quantity of Interest: Mean fraction of helical base-pairs (<Ph>):
– State j has a total of j helical base-pairs;
– Then, the fraction of helical base-pairs in state j:
• fj = j/N.
 As usual, a Weighted Average over all j yields Pb:
– From our Zipper model (Lecture 10):

– A plot of <Ph> vs. T approximates the DNA melting curve…

• Where Tm is the temperature where <Ph> = ½.
 Actual application requires definition of:
– A nucleation parameter, σ.
– A propagation parameter, s…which varies with T.
Defining the Propagation Parameter
 The parameter s defined for the forward transition:
– i.e., for the conversion from coil (c) to helix (h).
– s estimated by the Gibbs Factor:
s = exp[-∆Gcho/RT]
• where ∆Gcho = Goh-Goc is the free energy change:
– for the conversion of 1 residue from coil to helix.
– note: reference state is the all-coil state, C.
• i.e., the state of 0 free energy, and weight 1.
 Definition of s requires:
– consideration of the stabilizing energies in DNA.
– determination of the free energy change,

∆Gcho = ∆Hcho - T ∆Scho

• accompanying the transition of a residue from coil to helix.

 Helix Stabilized by Stacking
 DNA and RNA melting typically conceptualized:
– in terms of H-bond breakage:
• allowing strand separation…however:
 Nucleic acid helices stabilized primarily by stacking:
– Stacking b/w H-bonded base-pairs:
• hides hydrophobic rings from water;
• aligns ring dipole moments;
• maximizes ring VdW interactions;
• very favorable (<∆Hstacko> ~ -8.4 kcal/mol).
– In contrast, H-bonding b/w bases:
• only marginally (a little) favorable, in Aq. solution.
 Transition from c to h thus Exothermic:
– stacking of a base-pair releases heat…
• ∆Hcho = ∆Hstacko < 0.
Temperature-Dependence of s

 Since ∆Gcho is Temperature-dependent.

– s will also depend on T.
– An experimentally useful expression of this dependence
expressed by our Van’t Hoff relation:

 Since ∆Hoch < 0, we expect:

– for T < Tm, s > 1:
• propagation of a nucleated helix region favorable.
– for T > Tm, s < 1:
• propagation of a nucleated helix region inhibited.
 Sequence-Dependence of s

 In practice, stacking ∆Go’s depend on GC

content…
– And will vary with specific doublet identity:
• i.e., adjacent pairs of base-pairs.
– We will expect the size of our propagation
parameter:

s = exp[-∆Gcho/RT],

• to increase with GC content;

 Duplexes with higher GC-content:
– should form more easily…
• and be more resistant to melting.
 Sequence-dependence of Duplex Tm
 Increase in s with increasing GC content:
– due to increased stacking
favorability…
– observed experimentally by
variations in duplex Tm.
 Duplex Melting Temperature:
– increases roughly linearly with
increasing GC content.
– note that stacking is a very strong
interaction:
• most duplexes essentially completely stacked at 25o C.
 Stacking more favorable at high [Na+]:
– because of decreased electrostatic repulsion…
• due to counterion screening of the charged backbones.
 The Nucleation Parameter, σ
 The stacking process is cooperative:
– unstacking destabilizes neighboring, unmelted base-pairs.
• σ often estimated as follows…
 Unstacking a single, central base-pair:
– causes H-bond dissociation,
• with a weight change of ω’/ω = σ/s.
– Physically, this results in the loss of 2 stacks:
• 1 accounted for by 1/s.
• the other must be accounted for by σ.
 Thus, the cooperativity parameter, σ:
– can be estimated by the Gibbs factor:

σ = exp[∆Gostack/RT] = 1/s.

• experiments indicate a larger penalty: σ ~ 10-3-10-4.

– This justifies our Zipper approximation (since σ << 1).
Melting at the Middle vs. Ends
 Relative favorabilities of end and middle
melting:
– considered by comparing total statistical
weights:
• initiating melting at an end:
ωe = 2σsN-1
• initiating melting in the middle:
2 N-1
ωm = (N-2)σ s
• relative probabilities:
Pe/Pm = ωe/ωm = 2/Nσ.
 Similar to the result obtained for
polypeptides:
– ends melt first for duplexes with N < 2/σ;
• Short; high central GC-content.
– middle melts first for duplexes with N > 2/σ;
• Long; low central GC-content.
 DNA Annealing
 DNA Melting is kinetically simple:
– DNA begins as an intact, perfectly-aligned helix…
• melting progresses from the
ends or middle.
• shifted states very unlikely.
– Aligned Zipper model neglects:
• occupancy of ‘shifted’ states:
– assumes perfect alignment.
• conformations with internal loops.
• Reasonable for modeling the melting of short DNAs.
 The ‘reverse’ transition is DNA Annealing
– kinetically much more complex…
• single strands may nucleate in any alignment:
– need not be perfectly-aligned.
• thus, many other types of states may be occupied.
 Modeling DNA Annealing
 An adequate treatment of DNA Annealing:
– depends upon strand length, N.
 For short DNAs (N < 100 bps),
– a modified Zipper model may be adequate.
– such a model includes:
• concentration-dependence of nucleation.
• Frame-shifting (not shown) and hairpin formation.
– addressed in Lecture 13.
 For most DNAs (N>100):
– General model required:
• includes all structures.
– Q very complex:
• number of structures
scales ~ exp[N].
• not treated, here.
 Comparison with Experiment
 For short, quasirandom dsDNAs:
– Good agreement with DNA melting curve...
• Aligned Zipper Model (solid line).
• Experimental values (circles);
– Implication:
• A single cooperatively-melting region;
– As noted by the single sigmoid;
– Good agreement with annealing curve…
• Melting strictly reversible for short DNAs.
– But…NOT with fast cooling!
• Fast cooling causes Hysteresis (figure)…
– Curves distinct: indicates irreversibility.
 However - limited range of validity:
– Not valid for longer dsDNAs:
• 2 or more cooperatively-melting regions:
– Zipper approximation fails.
– Also inadequate for many short, annealing systems:
• e.g.: with staggered alignments/hairpin formation;
• Multi-strand systems (more than 2 ssDNA species).
– More general model: Lecture 12.
Transitions between Helices
 The ‘reference-state’ for dsDNA is a B-Helix,
– …under physiological conditions.
– however, DNA can adopt a variety of other helical structures:
• another slightly under-wound right-handed helix: A-DNA
– helical twist of about 32.7o vs. 34.3o (B-Helix)
• a left-handed helix: Z-DNA;
• a DNA triplex: H-DNA;
• DNA cruciforms, etc…
– Transitions between the various helices:
• have been observed in both linear and circular DNAs.

 Two of the interesting ones are:

– the transition from B-DNA to A-DNA (this lecture),
– the transition from B-DNA to Z-DNA (probably not covered)
 The B-DNA to A-DNA Transition
 The transition from B-DNA to A-DNA…
– can be induced in poly-[dG:dC] by addition of ethanol.
• as A-DNA is dehydrated, relative to B-DNA.
 This transition can be modeled
– Using the familiar aligned zipper model…
• By applying our expression for <Pb=A>:
– where <PA> is the mean fraction of base-pairs stacked in an
A-helical conformation.
– The propagation parameter, s is related to the Gibbs factor:
s = exp[-∆GoBA/RT] ,

• here, ∆GoBA = ∆GoA - ∆GoB, where:

∆GoB = free energy of stacking for a B-DNA base-pair doublet;
∆GoA = free energy of stacking for an A-DNA base-pair doublet.
– The nucleation parameter, σ = exp[-∆GoJ/RT] < 1.
• ∆GoJ = extra energy required to form two A-B junctions.
 Comparison with Experiment
 B-DNA to A-DNA transition in poly-[dG:dC]:
• induced by addition of ethanol.
– here, discrete points illustrate experimental data.
 Zipper model parameters:
– Propagation parameter, s:
• literature standard values.
– Nucleation parameter, σ = 0.14.
• transition only slightly
cooperative…
• due to a modest junction
energy :
∆GoJ = +5 kJ/mol.

– solid line indicates <PA>:

• excellent agreement.
Conclusion
 In this Lecture, we have discussed application of the
simple Zipper model:
– To the various transitions of Nucleic Acids:
• The DNA helix-coil transition (melting).
• DNA Annealing.
• The B-DNA to A-DNA transition.
– which demonstrate good experimental agreement

 Next lecture, we will extend our discussion of DNA

melting…
– By relaxing our assumption of perfectly-matched DNA.
• And discuss the ‘staggered zipper’ model.