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Artificial specific binders directly recovered from chemically modified nucleic acid libraries.

Kasahara Y, Kuwahara M - J Nucleic Acids (2012)

Bottom Line: 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.In addition, recent advances and possible future research are also described.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu 376-8515, Japan.

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.

No MeSH data available.


Examples of modified nucleoside triphosphates that act as substrates for polymerase reactions.
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Related In: Results  -  Collection


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fig2: Examples of modified nucleoside triphosphates that act as substrates for polymerase reactions.

Mentions: Among the modifications, DNA polymerases could endure substitution at the base moieties, and various functional groups could be introduced into those positions with relatively high efficiency. When artificially created protein-like functional nucleic acids were considered, researchers would first envisage introducing proteinous amino acids into nucleic acids. Indeed, base-modified dNTP analogs bearing various proteinous amino acids or their side chains have been reported to date, and their substrate properties in polymerase reactions such as PCR and primer extension have been investigated (Figure 2) [38]. For example, PCR assays using KOD Dash DNA polymerase showed that triphosphates containing amino acyl group with basic (Arg, His, Lys), aromatic (Phe, Trp), aliphatic (Leu, Pro), and neutral hydrophilic (Gln, Ser, Thr) side chains act as good substrates, while those with acidic (Asp, Glu) and thiol (Cys) side chains act as poor substrates. Production of DNA-containing cysteinyl residue necessitated the addition of dithiothreitol as a reduction reagent. To introduce plural functionalities with high density, it was found that four natural nucleotides (A, G, C, T) are totally replaced with four base-modified nucleotides by the addition of manganese chloride and betaine [32, 35]. Those additives could improve efficiency and yield in the enzymatic production of modified DNA, although they could raise the frequency of misincorporation at the same time [51, 52].


Artificial specific binders directly recovered from chemically modified nucleic acid libraries.

Kasahara Y, Kuwahara M - J Nucleic Acids (2012)

Examples of modified nucleoside triphosphates that act as substrates for polymerase reactions.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3472525&req=5

fig2: Examples of modified nucleoside triphosphates that act as substrates for polymerase reactions.
Mentions: Among the modifications, DNA polymerases could endure substitution at the base moieties, and various functional groups could be introduced into those positions with relatively high efficiency. When artificially created protein-like functional nucleic acids were considered, researchers would first envisage introducing proteinous amino acids into nucleic acids. Indeed, base-modified dNTP analogs bearing various proteinous amino acids or their side chains have been reported to date, and their substrate properties in polymerase reactions such as PCR and primer extension have been investigated (Figure 2) [38]. For example, PCR assays using KOD Dash DNA polymerase showed that triphosphates containing amino acyl group with basic (Arg, His, Lys), aromatic (Phe, Trp), aliphatic (Leu, Pro), and neutral hydrophilic (Gln, Ser, Thr) side chains act as good substrates, while those with acidic (Asp, Glu) and thiol (Cys) side chains act as poor substrates. Production of DNA-containing cysteinyl residue necessitated the addition of dithiothreitol as a reduction reagent. To introduce plural functionalities with high density, it was found that four natural nucleotides (A, G, C, T) are totally replaced with four base-modified nucleotides by the addition of manganese chloride and betaine [32, 35]. Those additives could improve efficiency and yield in the enzymatic production of modified DNA, although they could raise the frequency of misincorporation at the same time [51, 52].

Bottom Line: 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.In addition, recent advances and possible future research are also described.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu 376-8515, Japan.

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.

No MeSH data available.