Self-complementary RNA duplex melting

Self-complementary RNA duplex melting dissociates a duplex formed by two copies of the same RNA strand into separate strands. Self-complementarity means that the strand's reverse complement can match its own nucleotide sequence. The reaction has molecularity two in the association direction, unlike intramolecular RNA hairpin melting.

Use the ideal association model $2S\rightleftharpoons D$. Let total strand molar concentration be $c_T=[S]+2[D]$ and paired-strand fraction be $f=2[D]/c_T$. With reference molar concentration $c^{\circ }$, the dimensionless equilibrium constant isSubstituting $[S]=c_T(1-f)$ and $[D]=f{c}_T/2$ givesThe final expression is the physical solution for a self-complementary RNA duplex population; the algebraically equivalent subtractive form loses numerical precision at small $\alpha$.

At $\alpha =1$, half the strands are paired. For an original $c_T=10\mu \mathrm{M}$ example, this means $[D]=2.5\mu \mathrm{M}$ and $[S]=5\mu \mathrm{M}$. Duplexes are only one-third of the independently diffusing molecular objects, $[D]/([D]+[S])=1/3$. Half the strands is not half the objects. At fixed temperature and $K_a$, increasing $c_T$ tenfold makes $\alpha =10$ and $f=0.8$.

Heating changes $K_a$ and thus the population. At the strand-based midpoint, $K_a(T_m)=c^{\circ }/c_T$, which explains the concentration dependence of RNA melting. A fit should state whether its constant describes association or dissociation and whether concentration counts strands or duplexes.

Real sequences can also form competing hairpins. A primary tetraloop study explicitly compared predicted hairpin and duplex equilibria when designing its optical-melting sequences.^[ref] The equations above assume only separated strands and duplexes; they do not automatically cover competing folds or RNA melting hysteresis.

A self-complementary RNA duplex population describes how identical RNA strands are distributed between free strands and duplexes under specified conditions. A fraction of strands in duplexes differs from a fraction of molecular objects that are duplexes. The distinction matters when comparing chemical mass balances with observed signals.

For the ideal equilibrium $2S\rightleftharpoons D$, let $c_T=[S]+2[D]$ and $f=2[D]/c_T$. With dimensionless association constant $K_a$ and reference molar concentration $c^{\circ }$, define $\alpha =K_a{c}_T/c^{\circ }$. The strand balance givesThis is the population model underlying ideal self-complementary RNA duplex melting. It assumes separated strands and duplexes are the only relevant species and that chemical equilibrium is reached.

The concentration regimes are illuminating. For $\alpha \ll 1$, $f\approx 2\alpha$; for $\alpha \gg 1$, $1-f\approx 1/\sqrt{2\alpha }$. An original calculation gives $f\approx 0.0192$ at $\alpha =0.01$ and $f\approx 0.9317$ at $\alpha =100$. Even a large association parameter leaves some free strands in this equilibrium model.

An original finite-count illustration has 100 strands,80 of which belong to 40 duplexes, with 20 free strands left. The paired-strand fraction is 0.8, but duplexes are $40/(40+20)=2/3$ of the 60 molecular objects. A detector responding equally to each object would not give the same signal as a detector responding equally per strand. RNA optical melting needs a model of the folded and unfolded optical responses to infer populations.

Differentiating the equilibrium relation givesAt $f=0.5$, a small 10% relative change in $\alpha$ changes $f$ by approximately 0.0167. Population is therefore not generally proportional to concentration.

Competing RNA hairpins require an expanded species balance. A primary tetraloop study evaluated hairpin and duplex formation explicitly when designing sequences for melting experiments.^[ref] Ignoring a populated competitor can produce a precise solution to the wrong balance equation.