Expanding the Scope of T3 DNA Ligase-Catalyzed Oligonucleotide Polymerizations (T3-LOOPER)

Abstract

Antibodies are currently the gold standard in the field of therapeutics but are quite costly and chemically unstable. Nucleic acid polymers can mimic antibody therapeutics but are more cost effective to produce, can reversibly denature, and have a high synthesis reproducibility. These polymers are called ‘aptamers’. However, they lack in chemical diversity when compared to antibody-based therapeutics and are susceptible to renal filtration, making them inferior for specific binding. Chemical modifications can be attached to these DNA/RNA polymers to increase their diversity, though only four unique modifications can be incorporated into one polymer. Ligase-catalyzed oligonucleotide polymerization (LOOPER) is a method that synthesizes chemically modified nucleic acid libraries containing up to 16 unique chemical modifications. The method entails template directed ligations of chemically modified oligonucleotides. The purpose of this thesis is to push the boundaries of LOOPER to tolerate nucleobase modified oligonucleotides, sugar modified oligonucleotides, modification positioning on the oligonucleotide sequence, RNA oligonucleotides, and reverse transcription via LOOPER of a modified nucleic acid polymer back to its DNA form. A nucleobase modified library was synthesized to include predominantly hydrophobic modifications, allowing for an increased chance of developing an aptamer during in vitro selections against a protein target. The library generated a LOOPER yield of 19% with a fidelity of 94.8%, allowing this library to be a viable option for selections. When attempting LOOPER with sugar modified oligonucleotides, LOOPER was able to tolerate 2’-F at every position in the trinucleotide sequence and locked nucleic acids (LNA) at the middle position of the oligonucleotide. Fidelity analysis revealed that all 2’-F fidelities were over 90%. LNA oligonucleotide fidelities were unable to be characterized since the modification cannot be transcribed by commercially available polymerases. Luckily, we discovered that LOOPER can “reverse transcribe” the LNA-modified nucleic acid polymer back to DNA with a 99.0% fidelity. Lastly, the LOOPER system was not able to accurately ligate RNA oligonucleotides, as the fidelities were below 20%. LOOPER is a method that can “polymerize” and “reverse transcribe” modified nucleic acid polymers, making these libraries more accessible to the in vitro selection process.