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:

As usual, a Weighted Average over all j yields Pb:
– From our Zipper model (Lecture 10):

• fj = j/N.

– 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.

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