Artificial Specific Binders Directly Recovered from Chemically Modified Nucleic Acid Libraries

Abstract

Specific binders comprised of nucleic acids, that is, RNA/DNA aptamers, are attractive functional biopolymers owing to their potential broad application in medicine, food hygiene, environmental analysis, and biological research. Despite the large number of reports on selection of natural DNA/RNA aptamers, there are not many examples of direct screening of chemically modified nucleic acid aptamers. This is because of (i) the inferior efficiency and accuracy of polymerase reactions involving transcription/reverse-transcription of modified nucleotides compared with those of natural nucleotides, (ii) technical difficulties and additional time and effort required when using modified nucleic acid libraries, and (iii) ambiguous efficacies of chemical modifications in binding properties until recently; in contrast, the effects of chemical modifications on biostability are well studied using various nucleotide analogs. Although reports on the direct screening of a modified nucleic acid library remain in the minority, chemical modifications would be essential when further functional expansion of nucleic acid aptamers, in particular for medical and biological uses, is considered. This paper focuses on enzymatic production of chemically modified nucleic acids and their application to random screenings. In addition, recent advances and possible future research are also described.

Identification of a pKa-regulating motif stabilizing imidazole-modified double-stranded DNA

“…However, the innate absence of functional side chains in natural nucleic acids and the corresponding nucleotides poses clear limitations in the diversity of binders that can be obtained compared to protein-based ones. Examples exist that circumvent this issue through the introduction of modified residues thereby yielding new non-natural binders ( 16 – 19 ) and catalysts ( 20 – 24 ). However, the SELEX protocol becomes more labor intensive and reproducibility of results is often a problem in these cases.…”

Selection, Characterization and Application of Artificial DNA Aptamer Containing Appended Bases with Sub-nanomolar Affinity for a Salivary Biomarker

“…The amplification and replication of oligonucleotide strands selected by affinity separation with their target are key processes in the SELEX procedure. In particular, when modified DNA/RNA is used as a library 18 19 20 21 22 23 24 25 26 27 28 37 38 39 40 , the success or failure of the selection can greatly depend on the polymerase used, which affects the accuracy of modified nucleotide incorporation and the efficiency of modified oligonucleotides production 41 42 43 44 45 . In 2001, Sawai et al .…”

“…In 2001, Sawai et al . first discovered that KOD Dash DNA polymerase is one of the most promising candidates as a catalyst for enzymatic syntheses of modified DNAs 37 46 47 48 49 50 . Thereafter, KOD DNA polymerase-related products ( e.g.…”

In vitro selection of BNA (LNA) aptamers

“…This knowledge is now being widely accepted among researchers engaged in relevant studies. 54,[58][59][60]62,65,[79][80][81][82][83] To date, enzymatic syntheses of various modified DNAs using KOD-related DNA polymerases have been reported. A DNA polymerase named KOD XL is commonly used (which is essentially the same as KOD Dash); KOD XL comprises a mixture of approximately 2:98 wild-type KOD DNA polymerase and KOD(exo-) DNA polymerase.…”