Evolution of Functionally Enhanced α-l-Threofuranosyl Nucleic Acid Aptamers

Abstract

Synthetic genetic polymers (xeno-nucleic acids, XNAs) have the potential to transition aptamers from laboratory tools to therapeutic agents, but additional functionality is needed to compete with antibodies. Here, we describe the evolution of a biologically stable artificial genetic system composed of α-l-threofuranosyl nucleic acid (TNA) that facilitates the production of backbone- and base-modified aptamers termed “threomers” that function as high quality protein capture reagents. Threomers were discovered against two prototypical protein targets implicated in human diseases through a combination of in vitro selection and next-generation sequencing using uracil nucleotides that are uniformly equipped with aromatic side chains commonly found in the paratope of antibody–antigen crystal structures. Kinetic measurements reveal that the side chain modifications are critical for generating threomers with slow off-rate binding kinetics. These findings expand the chemical space of evolvable non-natural genetic systems to include functional groups that enhance protein target binding by mimicking the structural properties of traditional antibodies.

Nucleic Acids and Their Analogues for Biomedical Applications

“…The work demonstrated that TNA could fold into tertiary structures with retained chemical functions, suggesting TNA as an RNA progenitor in the pre-RNA world. After that, various TNA aptamers with the capability of binding to small molecules and large proteins have been selected [ 178 , 179 , 180 , 181 , 182 , 183 ]. These TNA aptamers have remarkable thermal and biological stability.…”

“…( a ) Schematic illumination of TNA aptamer targeting PD-L1 for cancer immunotherapy. Reprinted with permission from [ 183 ]. Copyright 2020, Royal Society of Chemistry.…”

From polymerase engineering to semi-synthetic life: artificial expansion of the central dogma

“…Different polymerases, including Therminator DNAP and a mutant of KOD DNAP, Kod-RI, have been used for the synthesis/transcription of TNA, and Bst DNAP has been used for the reverse transcription of TNA in these works. Recently, using TNA polymerase in combination with nucleobase-modified tNTPs, stable TNA aptamer with functionalized nucleobases have also been selected 202,203 . Mirror-image DNA has drawn broad interest in recent years, since it possesses good resistance to nucleases while retaining similar properties and functions of DNA 204 .…”

“Click handle”-modified 2′-deoxy-2′-fluoroarabino nucleic acid as a synthetic genetic polymer capable of post-polymerization functionalization

“…[4][5][6][7][8][9][10][11] To date, aptamers, XNAzymes, and nanostructures based on several different XNA systems have been reported, including 2 0 -deoxy-2 0 -uoroarabino nucleic acid (FANA), [12][13][14][15] hexitol nucleic acid (HNA), 16 locked nucleic acid (LNA), 17,18 and threose nucleic acid (TNA). [19][20][21][22] To enable the efficient uptake of XNA building blocks in the enzymatic polymerization reactions, engineered polymerases with altered substrate specicity were discovered through rational mutagenesis and directed evolution that can tolerate unnatural XNA nucleotide triphosphates (NTPs). For example, Holliger and coworkers evolved DNA polymerases that could recognize six classes of XNA NTPs including FANA, HNA, LNA, and TNA.…”