Lecture 12 – Structural Transitions in

Nucleic Acids II
 Outline
 Introduction
 The DNA Helix-Coil Transition
 Very quick review of basic DNA structure:
 Focus: base-pair stacking.
 DNA melting and melting curves:
 Thermal denaturation: breaking the stacks.
 Experimental monitoring of base-pair stacking %...

 Modeling DNA Melting:

 Idea: Generalize our Aligned Zipper model (Lecture 12).
 To treat concentration-dependence, shifted structures, loops…
 Focus is still: Equilibrium Treatment…
1. Weighting conformations (both stacks and loops);
2. Thermodynamic parameter sets;
3. Models of duplex formation;

 Comparisons with experiment.

 Our Focus: the Helix-Coil
Transition in DNA
 In particular, we focus on two
related processes:
 dsDNA melting
 B helix to two coils.
 dsDNA annealing
 two coils to a B helix.
 Note: single dsDNA species.
 Understanding these:
 aids in modeling more complicated
transitions.
 e.g., many competing species.
 Ultimate focus: complex annealing.
 Single-Stranded DNA (the ‘Coil’)
 An unbranched, polynucleotide chain:
 monomers units = nucleotides.
 each contains three components:
a negatively charged phosphate (PO4-);
a 2’-deoxyribose sugar;
one of 4 heterocyclic bases (A,T,G,C);
 pairs linked by phosphodiester bonds.
 Nitrogenous bases are of two types:
 Purines (2 rings): Adenine (A), Guanine (G).
 Pyrimidines (1 ring): Thymine (T), Cytosine (C).
 ssDNA has a 5’ to 3’ polarity.
 5’ and 3’ ends are chemically distinct.
 by convention, DNA sequence is written 5’ to 3’.
 The B Helix
 Natural dsDNAs in solution…
 adopt a double-stranded, helical structure.
 strand orientations are antiparallel.
 under physiological conditions: B helix.
 Helix characterized by Watson-Crick
base pairing:
 A pairs with T (2 H+-bonds).
 G pairs with C (3 H+-bonds).
 At right, we show the dsDNA formed
by annealing of:
 5’-CTAGTCGTGGTTC-3’
 5’-GAACCACGACTAG-3’
 Stabilizing Interactions

 DNA B-Helix structure stabilized by:

 Hydrogen bonding between bases (minor);
 Stacking between H+-bonded base-pairs (primary):
1. Van der Waals interaction:
 Optimal stacking of adjacent rings;
2. Induced dipole-induced dipole interaction:
 Favorable alignment of the moments of adjacent rings.
 Results in the characteristic helix shape.
3. Hydrophobic interaction:
 Favorable sequestering of hydrophobic rings.
 In DNA melting…the helix destabilized
 generally implemented by increasing temperature, T.
 destabilizes the stacks…unwinding the helix.
 unwound helix separates into free ssDNAs (‘coils’).
 Monitoring the Helix-Coil
Transition
 Degree of stacking is experimentally observable:
 Let ΘB = mean fraction of stacked base pairs.
 Ultraviolet absorbance at 260 nm (A260)…
is inversely proportional to ΘB;
Also called the ‘hypochromicity’.
 DNA melting accompanied by  40% increase in A260.
 Plot of A260 vs. T yields ΘB vs. T
 The DNA ‘melting curve’.
 DNA Melting Curves
 ΘB decreases monotonically from 1 to 0 (for fully matched strands).
 sigmoidal shape indicates DNA melting is cooperative.
 One sigmoid = cooperative melting of entire DNA;
 The DNA melting temperature (Tm):
 For fully matching strands: temp. at which ΘB = ½;
 Width (∆T) is non-zero (e.g., for 10-mers, ∆T  10 oC).

 Melting curves of longer DNAs show more structure:

 several independently melting regions (AT’s less stable).
 Why focus on DNA Melting?
 Fitting of model curves with experiment:
 facilitates investigation of DNA thermodynamic parameters.
 describe the fundamental properties of DNA interaction.
 Helps to understand more general DNA mixtures.

 Modeling provides a demonstration of general techniques:

 Equilibrium chemistry;
 Statistical Thermodynamic weighting;

 Although parameter values vary by polymer, transition,

 general principles apply to modeling other biopolymers:
 protein folding and structural transitions.
 RNA folding, etc.

 We will use an Equilibrium approach…

 based on Statistical Thermodynamics.
 The Aligned-Zipper Model
 In L. 11, we adopted a simplified model…
 Three non-zipper assumptions:
 Homoduplex-melting:1 kind of base-pair.
 Strands perfectly-aligned: no shifting.
 Strand-separation neglected: no [strand] effects.

 This allowed an aligned Zipper model:

 Annealing: forward transition (coil to helix).
 Melting: reverse transition (helix to coil).
 from a fully-helical state, H = …hhhhhh….
 to a fully-melted state, C = …cccccc….

 Model Application proceeded by:

1. Defining model parameters:
 The nucleation parameter, σ.
 The propagation parameter, s.
 Applying our Zipper-expression for <Ph>:
 Result: DNA Melting Curve.

 However, our model a bit too simple!

 Need for a Better Model
 Most dsDNAs of interest are not homo-polymers:
 Generally contain all 4 bases (A, T, G, C).
 At least 10 propagation parameters, si required.

 Strand-separation is also significant:

 Results in a dependence on strand-concentration.
 Particularly for oligonucleotides.

 Annealing, Melting also much more complicated:

 Shifted alignments, looped structures can be significant.
 Melting Curve Prediction:
 We adopt an equilibrium model.
 Our simple equilibrium of interest:
 Quantities of Interest:
 Fraction of stacked base pairs (bps): ΘB = ΘextΘint;
 fraction of associated strands: Θext = 2CAB/Ctot;
 mean fraction of stacked bps per dsDNA conformation: Θint ;

 Let’s begin with an estimation of Θext

 First, we need some simple equations:
 Mass Action: KD = CACB /CAB = 1/Kassoc.
 ssDNA Strand Conservation:
 CAo = CA + CAB and CBo= CB + CAB
 Melting Curve Prediction (cont):

 Continuing our estimation of Θext

 Analysis is system-dependent:
 Mass Action: KD = CACB /CAB = 1/Kassoc.
 ssDNA Strand Conservation:
 CAo = CA + CAB and CBo= CB + CAB

 Idea: Combine to yield a quadratic eq. ( solve for Θext…)

 Usual is an ‘Equivalent co-polymer’ treatment: assume A = B;
 Good for long polymers; not so good for oligonucleotides.

 Result: Θext = [(Ctot/KD+1) - (1+2Ctot/KD)1/2] / (Ctot/KD)

 Statistical Thermodynamics
 Computing ΘB still requires estimates of KD and Θint.
 Tool…Statistical Thermodynamics.
 assumption: system always (nearly) at equilibrium.
 note a limitation…no rate information.
 Consider an Ensemble of Systems:
 large number of instances of our system…O(1023).
each prepared identically.
 members distributed over all accessible conformations:
single-stranded states (unstacked ssDNAs, hairpins).
distinct double-stranded states.
 Stat-thermo addresses:
 equilibrium probabilities of state occupancy.
 changes in system variables which accompany equilibrium
transitions.
 Ising Model of Stacking
 Assumption: stack-formation is all-or-none.
 each base has either single-stranded or stacked ‘character’.
 big simplification…

 Each dsDNA conformation is then specified by:

 alignment between the interaction strands.
 stacking pattern.
 no worrying about “partial” stacks.

 Conformation specified by location of helical and ss regions.

 The Gibbs Factor
 So, how do we estimate relative occupancies?

 As before, each conformation, i gets a statistical weight, ωi.

 related to its standard free energy of formation, ∆Gio:
 ωi = exp[-∆Gio/RT]; R = molar gas constant.
 the ‘Gibbs Factor’.

 Relative probability of observing states i and j:

 estimated by the ratio of weights:
P(i)/P(j) = ωi/ωj = exp[-δ(∆G)o/RT]…
 at equilibrium, more stable states much more likely.

 What about the absolute probabilities?

 we need to normalize by dividing by the Partition Function…
 The Partition Function, Z
 Z = the sum of the statistical weights of all states:

Z = Σi ωi = Σi exp[-∆Gio/RT]

 (we called this ‘Q’, earlier…)

 equal to the product of external and internal Z’s:
Z = ZextZint.

 As before, the absolute probability of observing any state, i:

P(i) = ωi/Z.

 All thermodynamic quantities derivable from Z.

 macroscopic observables correspond to ensemble averages.
 ensemble average <X> of observable X:
<X> = Σi Xi ωi / Z;
Xi = X value characteristic of state i.
 Estimating KD
 KD = equilibrium constant of dissociation.
 estimated by the partition functions of products and reactants.
 ‘reactants’ = all double-strands (dsDNAs).
 ‘products’ = all fully melted single strands (ssDNAs).

 For dsDNA melting: KD = Z(ss)2 /Z(ds)=1/(βZc).

 Zc = ratio of internal partition functions = Zint(ds).
 β = ratio of external partition functions = Zext(ds)/Zext2(ss).
= the ‘strand association parameter’.

 Note: If we like, we can also model hairpin melting:

KD = Z(ss)/Z(hp) = 1/Zc.

 Also note that KD = 1/Kassoc.

 Estimating Θint
 Recall that ΘB = Θint Θext.
 KD allows us to model Θext.
 however, Θint must also be estimated…
The mean fraction of stacked bps/duplex.
 Let fi denote the fraction of stacked base pairs in
conformation i.
 then, Θint is just the ensemble average of fi…
 denoted, <fi>.
 Θint can be estimated from the partition function:

Θint = <fi> = Σi fiωi/Zc

 Here, Zc = Zint is the conformal partition function;

 Only the weight of associated conformations included.

.
 Estimating Statistical Weights
 Now we know how to compute Θint and Θext.
 We also have: Kassoc = βZc = β Σi ωi…
 How do we estimate the statistical weight, ωi of each
conformation?
 by decomposition.
 We model a given dsDNA conformation X…
 a ‘linear’ chain, consisting of simpler structures:
 base-pair doublets, hairpin loops, internal loops, bulges…
 each weighted independently…
 weights form a set of thermodynamic parameters.
 product of weights = overall weight.
 Example
 Decomposition of a conformation into subunits…

 Overall Statistical Weight

 product of a set of smaller weights…
 which are determined by the identities the subunits.
 Statistical Weight (cont.)

 Overall weight of conformation X denoted ωX.

 ωx estimated by a product of smaller weights.

 Distinct weight for each type of structure…

 si - each stacked base pair doublet of type i.
 σ1/2 - each junction between stacked/unstacked pairs.
 f(m) - each internal loop of m broken base pairs.
 sbulge - each bulge loop.
 F(n) - each terminal (hairpin) loop of n bases.
 send- each dangling end.
 we must also assign a weight for dsDNA chain association…β.
 We now address each, in turn.
 Statistical Weight of a Stacked
Base-pair Doublet, s
 Nearest-neighbor model:
 Enthalpy (∆Ho) and Entropy (∆So) of doublet stack formation.
 depend only on the identity of the base pair doublet.
 10 types of Watson-Crick base pairs = 20 distinct parameters.
 Statistical weight of a stacked base-pair:
 snn(i) = exp[-∆Gio/RT].
 ∆Gio = ∆Hio–T∆Sio = Gibbs free energy of stacking.
 Many Nearest-neighbor parameter sets:
 10 Watson-Crick pairs (SantaLucia, et al., 1998).
 Various singlet-mismatches (Allawi, et al., 1997, etc.).
 Example:
 Sequence-Dependence of s
 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.
 Modeling End Unraveling:
The Cooperativity Parameter (σ)
 Unraveling at a duplex end:
 generally modeled by σ1/2.
 σ accounts for the cooperativity of DNA melting.
 formation of an isolated base much more difficult.
 Consensus value: σ = 4.5 x 10-5 (0.1 M [Na+]).
 Some care is required:
 σ always included in chain association parameter, β.
 σ1/2 sometimes included in terminal loop weight, F(m).
 1 factor of σ1/2 sometimes ‘normalized’ into the zero free
energy state (Benight, et al., 1988).
 σ is also salt-dependent (S. Kozyavkin, 1987).
 Statistical Weight of an Internal Loop
 Internal loop of m unbonded base pairs:
 Statistical weight = σfn(m):

 End unraveling:
 accounted for by 2 factors of σ1/2.
 Normalized probability of loop closure:
 Jacobson-Stockmayer: f(n) = 1/n1.5; unrestricted loop, n links.
 Purely entropic in origin (no T-dependence).
 Empirical form (R. Wartell, 1977)
 f(m) = 1/[(1-1.38-0.1m)(m+1)1.7], m > 3.
 Accounts for volume exclusion and chain stiffness.
 Note: Due to the large penalty…
 Looped conformations usually discarded for oligos.
 Statistical Weight of a Bulge Loop
 Bulge Loop
 only one strand has unpaired bases.
 Example:

 Perturbation to intact helix, δ∆Go.

 statistical weight, sbulge = exp[-δ∆Go/RT]
 1-base bulges well-studied (Zhu and Wartell, 1999):
 statistical penalty roughly σ1/2 ; sequence-dependent.
 Larger bulges less well-studied:
 parameters for RNA bulges (Freier, et al., 1986).
 statistical penalty > σ, increases with size.
 Statistical Weight of a Terminal Loop
 A terminal loop of n unpaired bases:
 “hairpin” loop.
 Statistical weight = σ1/2Fend(n).
 Example:

 Strand unraveling:
 modeled by σ1/2.
 Normalized probability of loop closure:
 Fend(n) = M(n)/(n+1)1.5 (Benight, et al., 1988).
 M(n) accounts for steric hindrance, chain stiffness.
 Statistical Weight of a Dangling End
 Dangling ends (overhanging, unpaired bases):
 stacking of first dangling base against duplex core.
 often as stabilizing as an extra stack.

 Energetics sequence dependent.

 Nearest-neighbor model, δ∆Go (Bommarito, et al., 2000).
 Energies depend, to 1st order only on:
 Identity of dangling base + duplex core bases;
 Statistical weight, sdangle = exp[-δ∆Go/RT].
 Values for all dangles reported.
 Bimolecular Helix Initiation
 Strand Association Parameter: β.

 β Accounts for both nucleation and end unraveling.

 includes a factor of σ (one σ1/2 for each duplex end).
 Length, temperature dependent (W. Hillen, 1981).
 β = KNa+b[1-Θ(int)]; K = 5000, a = -2.8, b = -3.2 ([Na+] = 0.1 M).
 Nearest-neighbor model (SantaLucia, et al., 1998):
 Simpler, approximate treatment of β (deviations < 20%).
 length-independent “initiation” free energy, ∆Gonuc.
 β = exp[-∆Gonuc/RT].
 Impact of Strand Anchorage

 For duplex conformations formed on microchips

 well known to be much less stable.

 Impact may be treated as a multiplicative correction:

 A. Fotin, et al., 1998.
 length, nature of linker – no substantial effect.
 δ∆Ho = 24 +/- 4 kcal/mol
 δ∆So = 70 +/- 12 cal/(mol K)
 Then, δ∆Go = δ∆Ho – T δ∆So
 sanchorage = exp[-δ∆Go/RT].
 Example 1 – Simple DNA Duplex

 Kd for formation from isolated strands:

 one factor of β for helix initiation.
 an appropriate factor of s for each stacked base pair.
 recall internal partition function for each isolated strand = 1.
 Kd = 1/βsCC/GG2sCA/GTsAA/TT2 = 1/Ka.
 Approx. form for a conformation of this type:
 let s = mean weight of doublet stacking.
 Kd = 1/βsl-1.
 Example 2 – Simple DNA Hairpin

 Kd for formation from unfolded single-strand:

 one factor of ‘σ1/2F(7)’, for the terminal loop.
 one factor of σ1/2, for unraveling at the free end.
 an appropriate factor of s for each stacked base-pair.
 recall internal partition function for the isolated strand = 1.
 Ka = σsCC/GG2sCA/GTsAA/TT2 F(7); then Kd = 1/Ka.
 Approx. form for a conformation of this type:
 let s = mean weight of doublet stacking.
 Ka = σsl-1M(n)/(n+1)1.5.
 Models of Duplex Formation

 General Model = all conformations considered.

 Problem: Exponential number of statistical weights.
computationally too intensive.
 Solution: choose a simpler model of duplex formation.
various models have been investigated…
 Models of Duplex Formation (cont.)
 All-or-None Model (2 states):
 full-length duplex and dissociated ssDNA pair.
 good approximation for KD (complementary strands);
 Appropriate for short oligos (< 15 bps);
 Fully Aligned Model (no shifted alignments).
 still an exponential number of states, but…
 Z can be computed in TIME O(N2)…Poland’s Algorithm.
 appropriate for DNA Melting (all lengths).
 Staggered Zipper Model (1 duplex/conformation):
 includes all shifted alignments; no internal loops larger than 1.

 Z computable in TIME = O(N2).

 With singlet bulges, TIME = O(N3).
 appropriate for annealing/renaturation of short DNA.
limit: N < 100-150 bps.
 Simplest Application: Short Oligos
 For short oligos, a 2-state model often used;
 Only 2 conformations:
 un-melted (2 ssDNAs) + fully-aligned dsDNA;
 Applied model can be statistical (melting curves), or van’t Hoff.
 Generally, focus is Tm value…
 Van’t Hoff assumption: @ Tm, Θ = Θ(ext) = ½.
 Example: Length 9 bp mis-matched oligo:

 Result: All-or-none model gives good agreement for short oligos:

 Both ∆Go and Tm predictions are acceptable (SantaLucia, et al.);
 However: for oligos longer than 15 bps, a shifted Zipper model required...
 Limitations of the 2-state Model
 Is a 2-state model good for long oligonucleotides?
 Study: 100 dsDNAs of length 23 bps (A. Suyama, et al).
 Experimental vs. Calculated Tm values:

 Result:
 correlation pretty good, but...calculated values too low!
 Unpredicted stabilization probably due to melting intermediates.
 Adequate for predicting gross behavior/trends;
 Inadequate for accurate or detailed prediction.
 Longer DNAs
 Statistical Zipper Model (1 duplex/conformation):
 very successful for predicting DNA melting, up to 150 bps;
 e.g.: Differential melting curves for 4 lac DNA fragments:
 Watson-Crick SZM predictions shown;
 Note add’l structure: 2 melting regions (two peaks) in (d)…
 For polymers > 150, a general, aligned model usually required.

a: 80 bps,
b: 101 bps,
c: 188 bps,
d: 219 bps
experimental.
---- theoretical.
 Conclusion
In this Lecture, we have:
 Discussed the Helix-Coil transition of biopolymers.
in the context of DNA melting and renaturation.
 Described physical methods necessary for modeling:
Equilibrium Chemistry and Statistical Thermodynamics.
Note: also apply to protein and polysaccharide modeling.
 Investigated the generalization of the model:
 DNA strand design:
 Stat-thermo modeling of error/efficiency.
 Quantitative design for minimized error.

Next come Real-world applications:

 Low-error Tag-Antitag system design.