Generation of Synthetic Copolymer Libraries by Combinatorial Assembly on Nucleic Acid Templates

Abstract

Recent advances in nucleic acid-templated copolymerization have expanded the scope of sequence-controlled synthetic copolymers beyond the molecular architectures witnessed in nature. This has enabled the power of molecular evolution to be applied to synthetic copolymer libraries to evolve molecular function ranging from molecular recognition to catalysis. This Review seeks to summarize different approaches available to generate sequence-defined monodispersed synthetic copolymer libraries using nucleic acid-templated polymerization. Key concepts and principles governing nucleic acid-templated polymerization, as well as the fidelity of various copolymerization technologies, will be described. The Review will focus on methods that enable the combinatorial generation of copolymer libraries and their molecular evolution for desired function.

Evolutionary Outcomes of Diversely Functionalized Aptamers Isolated from in Vitro Evolution

“…LOOPER exhibits an uneven incorporation of modifications resulting from biases in polymerization. Incorporation of a given modification is affected by the GC content of its codon and compatibility with T4 DNA ligase . LOOPER codon enrichment is calculated as the frequency of a codon in the polymer divided by the frequency of the same codon in the template.…”

“…Current strategies to produce unnatural nucleic acid polymers can be broadly classified into three categories: (i) synthesis with natural nucleotide triphosphates modified at the phosphate, , sugar ring, − or nucleobase; , (ii) synthesis with an expanded genetic code; − and (iii) postsynthesis modification using click chemistry. , More recently, we developed the ligase-catalyzed oligonucleotide polymerization (LOOPER) to further expand aptamer functionality (Figure a). − This method produces sequence-defined diversely functionalized nucleic acids libraries by ligating functionalized pentanucleotide anticodons across from cognate codons in a DNA-templated manner. To date, the largest codon set that has been used in LOOPER is defined as XXNNT (anticodon defined as ANNXX), where XX is a specific dinucleotide that encodes for one of 16 possible modifications on the cognate anticodon, whereas NN is a variable dinucleotide sequence that does not encode for the modification, but provides degeneracy (Figure b).…”

Expanding the Chemical Diversity of DNA

“…By using this approach, a vast number of functionalized nucleic acid polymers with binding or catalytic properties have been discovered. 10,14 Importantly, the expansion of chemical diversity within the evolvable nucleic acid polymer allows access to better binding kinetics against molecular targets 15 and enhanced catalytic activities. 16,17 Notwithstanding, the polymerase-based approach has a key shortcoming, which ostensibly limits its ability to access the level of chemical diversity present in proteins; namely, that the number of unique modifications is limited by the number of nucleobases in the genetic code, which is limited to four in a standard genetic code.…”

“…Chemists have long realized the potential of incorporating functional diversity throughout a nucleic acid polymer to enhance its functional potential and bridge the gap between nucleic acids and proteins. 10 As a polymer with predictable and reversible folding properties, nucleic acid polymers decorated with the rich chemical diversity of proteins could have enormous potential in fields ranging from medical diagnostics to molecular catalysis. However, robust methods to rapidly generate and evolve libraries of these envisaged polymers have only recently become available.…”

Structure–activity relationships of the ATP cofactor in ligase-catalysed oligonucleotide polymerisations

“…To address the functional group deficit of aptamers, researchers have explored ways to sequence-specifically incorporate diverse functionality either through nucleobase or sugar backbone modifications. 6–7 Indeed, enhancing the functional group repertoire of nucleic acid polymers has yielded more favourable binding properties, 8 and also expanded the utility of this class of polymer within the realm of catalysis. 9–11 …”