Reversible Switching of Cooperating Replicators

How can molecules with short lifetimes preserve their information over millions of years? For evolution to occur, information-carrying molecules have to replicate before they degrade. Our experiments reveal a robust, reversible cooperation mechanism in oligonucleotide replication. Two inherently slow replicating hairpin molecules can transfer their information to fast crossbreed replicators that outgrow the hairpins. The reverse is also possible. When one replication initiation site is missing, single hairpins reemerge from the crossbreed. With this mechanism, interacting replicators can switch between the hairpin and crossbreed mode, revealing a flexible adaptation to different boundary conditions.

Figure 1

Three modes of replication: linear, hairpin, and crossbreed. A linear oligonucleotide with one initiation site leads to linear product increase during PCR (blue circles). Hairpins replicate exponentially with a single initiation site. The secondary structure slows replication by self-inhibition (red squares). Linear DNA replicates exponentially if two initiation sites are present (red-blue triangles). Arrows indicate transitions between replication modes. (b) Degradation is implemented by serial dilutions. After $n$ replication rounds, the replicated DNA is transferred to $d-1$ parts of fresh replication medium ($n=6$; $d=5$). Linear replicators (left) vanish. Exponential replicators (middle, right) survive if the replication rate exceeds the dilution rate. Dashed curves are exponential fits, gray curves show the theoretical dynamics between the dilutions [Eqs. (1)–(3)].

Figure 2

Crossbreeding and hairpin regrowth. (a) Two hairpins with same loop but different initiation sites cooperate to form crossbreeds. Replication is interrupted and an incomplete strand emerges. It cross-hybridizes to another hairpin and replication is completed (upper pathway). Two crossbreed variants are possible, differing by a sequence inversion in their center. The inversion allows hairpins to regrow: The replication is again interrupted and completed after cross-hybridization (lower pathway). The processes can equally well start with the species marked by $*$ or $**$. (b) Different lengths and fluorophores (Cy3, Cy5) allow us to distinguish the species in gel electrophoresis. The replication of cooperating hairpins leads to crossbreeds (top). Starting from crossbreeds with one active initiation site the hairpins regrow (bottom). Both processes work for a wide range of annealing temperatures. First and last lanes are Cy5-labeled 84-mers as length reference.

Figure 3

Crossbreeding and hairpin regrowth under serial dilution. (a) Two hairpins with the same loop sequence go extinct under dilution conditions with $n=6$ and $d=5$ (left). However, the hairpins form crossbreeds, which slowly emerge [$\beta =4.5\times {10}^{-5}{\mathrm{nM}}^{-1}$, see Eqs. (4) and (5)] and take over the population. Hairpins with nonmatching orthogonal loop sequences vanish after a few cycles and no crossbreeds emerge (right, $\beta =0{\mathrm{nM}}^{-1}$). (b) Linearly replicating crossbreeds go extinct under milder dilutions ($n=15$; $d=2$). When both crossbreed variants are present, the sequence inversion allows hairpin regrowth (left, $\alpha =3.2\times {10}^{-4}{\mathrm{nM}}^{-1}$). Otherwise, all information is lost (right, $\alpha =0{\mathrm{nM}}^{-1}$).

Figure 4

Surviving an alternating number of initiation sites (blue and red-black). A simulation of hairpin replicators under changing environmental conditions shows how hairpins benefit from crossbreeding. Here, the dilution is implemented after every cycle ($n=1$; $d=1.18$) and the initiation sites are switched on and off periodically with a phase shift. Left: When both initiation sites are active (gray bars), the hairpins replicate and form exponentially replicating crossbreeds ($\beta =4.5\times {10}^{-5}{\mathrm{nM}}^{-1}$). With a missing initiation site, only one hairpin replicates, crossbreeds replicate linearly and regrow the hairpins ($\alpha =3.2\times {10}^{-4}{\mathrm{nM}}^{-1}$). Crossbreeding and hairpin regrowth allow the hairpins to maintain their sequence information. Right: Without crossbreeding the hairpins vanish ($\alpha =\beta =0{\mathrm{nM}}^{-1}$).