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Abstract
Aptamers, single-stranded nucleic acid polymers, are known to bind specific targets with high affinity and specificity. However, functionalities of natural aptamers are limited by four canonical DNA bases compared with 20 amino acids. This deficit of nucleic acids makes it difficult for aptamers to match the performance of antibodies as affinity reagents. Traditional incorporation of chemical functionality has relied on the individual modified nucleobases, which limits the number of unique modifications up to four. T4 DNA ligase-catalyzed oligonucleotide polymerization (LOOPER) has the ability to expand this limitation by catalyzing the ligation of modified 5-phosphorylated oligonucleotides on a single-stranded DNA template. Thus, the number of unique modifications relies on the codons rather than single nucleotides. Hexylamine modified adenosine is the chemical handle to introduce various functionalities to the natural nucleic acid polymers through amide bond formation. The high fidelity of LOOPER system has been confirmed by Duplex Sequencing method.Different functional groups such as hydroxyl groups and aromatic rings are successfully incorporated on the hexylamine modified ANNNN building block libraries. These homofunctionalized ANNNN libraries have high polymerization (>60%) and fidelity (>90%), as well as low codon bias. The LOOPER system retains high efficiency and fidelity when applied to a complex heterofunctionalized system: 16 sub-libraries with different modifications. Thus, LOOPER system enables the generation of diversely functionalized nucleic acid polymers. Current efforts are being directed to the in vitro selection of highly modified aptamers against glycosaminoglycans.