Mechanical breeding of DNA/RNA sequences

The sequence-function relation in biology is uni-directional. Sequences are rejected in molecular evolution if they do not show a desired function. On the other hand, when molecular functions are designed by scientists, this logic is inverted: a function is designed by trial and error. Subsequent breeding of function by Selex is still limited by sequence space and selection protocols. Therefore, the feedback loop between theory-based molecular design and functional tests is only within a given sequence pool.

We will explore an autonomous strategy that combines both paradigms. Without human intervention on the level of molecules, the functions of the molecules will be selected by a mechanical, microfluidic parkour under the amplification of a cooperative phase transition. This approach is motivated by the preliminary finding that random sequences of RNA are selected solely on the basis of Earth's gravitation. Matching sequences form hydrogels and become so large and heavy that they sediment within less than an hour. We will explore the possibilities of such a purely mechanical selection of functional sequences and create microfluidic flow settings that autonomously select sequences from random RNA or DNA based on their macroscopic binding phenotype. For example, we will select a phenotype of small or large grained DNA/RNA gels, slow or fast sedimenting and weak or strong binding DNA/RNA. A special focus will be to select purely by gravity the RNA aptamers forming gels only upon binding to a target molecule i.e. a gelation co-factor. We will target the binding of RNA to short peptides.
Previously, we have observed that apart from gravity, the movement in a temperature gradient is a strong selection force and can lead to complex molecular interaction networks. We have already shown that pools of tRNA-like molecules form gels in thermal traps or water-gas interfaces and implement a complex replicative function using simple chemistry and helped by salt cycling from physical phase transitions. In preliminary ligation experiments we have seen that temperature cycling leads to non-trivial sequence selection and a symmetry-breaking dynamics in sequence space with defined sequences. Both let us assume that a complex cooperative sequence dynamic is to be expected from the above proposed gel selection experiments. We have experience with chemical ligation and managed to tune Illumina sequencing for very short sequences of only 12mers. Sequencing will be our preferred readout method for analysis together with fluorescence imaging. We expect a rich cooperative sequence pattern that evolves over subsequent cycles of mechanical selection.
The natural connection with DNA origami approaches will connect our experiments with various groups in the SFB (Simmel (A02), Liedl (A06). Existing cooperation on the theory level with the Gerland (A03) and Frey (B02) groups will be expanded. We will share within the SFB our expertise of sequencing and affinity measurement in complex situations with microscale thermophoresis.