In Vitro Selection of Diversely Functionalized Aptamers

Abstract

We describe the application of T4 DNA ligase-catalyzed DNA templated oligonucleotide polymerization toward the evolution of a diversely functionalized nucleic acid aptamer for human α-thrombin. Using a 256-membered ANNNN comonomer library comprising 16 sublibraries modified with different functional groups, a highly functionalized aptamer for thrombin was raised with a dissociation constant of 1.6 nM. The aptamer was found to be selective for thrombin and required the modifications for binding affinity. This study demonstrates the most differentially functionalized nucleic acid aptamer discovered by in vitro selection and should enable the future exploration of functional group dependence during the evolution of nucleic acid polymer activity.

Influence of Linker Length on Ligase‐Catalyzed Oligonucleotide Polymerization

“…Thus, when encoding for an alcohol modification, XX=AA, AT, TA, and TT should be avoided. However, we have successfully encoded problematic alcohol modifications with XX=GG with excellent fidelities …”

“…Expansion beyond four modifications requires an alternative strategy that does not rely on polymerase‐mediated incorporation. To this end, Ligase‐catalyzed OligOnucleotide PolymERization (LOOPER) permits the expansion of chemical diversity in DNA beyond four different modifications (Scheme ) . LOOPER involves the copolymerization, catalyzed by T4 DNA ligase, of a library of modified oligonucleotides along a library of DNA templates.…”

Biotechnology Tools Derived from the Bacteriophage/Bacteria Arms Race

“…Ligase in vitro activity, particularly its ability to accept modified ligands, has been extensively explored for the assembly of heavily modified DNA sequences for aptamer selection [49,50] and to explore a wider range of nucleic acid modifications, such as sugar-modified nucleic acids [51].…”

“…DNA modifications, particularly modifications that bring chemical functionality not available in natural bases, such as glycosylation in Bacillus subtilis SP-15 phage [76], can be harnessed for function as has been achieved through the chemical modification of DNA bases and systematic evolution of ligands by exponential enrichment (SELEX) [50,77]. Despite characterisation of the biosynthetic pathway for multiple-phage DNA modification systems, none have been implemented in vitro for applications.…”

XNA ligation using T4 DNA ligase in crowding conditions

“…This strategy has been used for the generation of highly functionalized base-modified DNA aptamers. 11,12 T4 DNA ligase catalyses the phosphodiester bond formation between the 3′-hydroxyl group ('acceptor') and the 5′-phosphate terminus ('donor') of juxtaposed oligonucleotides in nicked DNA or, in some cases, in a hybrid DNA/RNA or RNA duplex. [13][14][15][16][17] Additionally, it can join blunt and cohesive ends.…”