Expanding the chemical diversity of aptamers remains an important thrust in the field in order to increase their functional potential. Previously, our group developed LOOPER, which enables the incorporation of up to 16 unique modifications throughout a ssDNA sequence, and applied it to the in vitro evolution of thrombin binders. As LOOPER-derived highly modified nucleic acids polymers are governed by two interrelated evolutionary variables, namely, functional modifications and sequence, the evolution of this polymer contrasts with that of canonical DNA. Herein we provide in-depth analysis of the evolution, including structure-activity relationships, mapping of evolutionary pressures on the library, and analysis of plausible evolutionary pathways that resulted in the first LOOPER-derived aptamer, TBL1. A detailed picture of how TBL1 interacts with thrombin and how it may mimic known peptide binders of thrombin is also proposed. Structural modeling and folding studies afford insights into how the aptamer displays critical modifications and also how modifications enhance the structural stability of the aptamer. A discussion of benefits and potential limitations of LOOPER during in vitro evolution is provided, which will serve to guide future evolutions of this highly modified class of aptamers.
Keywords: DNA-encoding; ligase-catalyzed oligonucleotide polymerization (LOOPER); sequence-defined polymers; unnatural nucleic acid aptamers.