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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.


Preparation scheme for chemically modified nucleic acid aptamers that bind to VEGF. High nuclease-resistant 2′-methoxy nucleotides were introduced through Post-SELEX modification process (left), and fully modified 2′-OMe RNA aptamers were directly selected from a library of 2′-OMe transcripts (right).
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fig1: Preparation scheme for chemically modified nucleic acid aptamers that bind to VEGF. High nuclease-resistant 2′-methoxy nucleotides were introduced through Post-SELEX modification process (left), and fully modified 2′-OMe RNA aptamers were directly selected from a library of 2′-OMe transcripts (right).

Mentions: Owing to the limited tolerance for modified substrates of the RNA polymerase (T7 RNA polymerase) used for SELEX, the 2′-methoxy (–OMe) groups need to be replaced with 2′-hydroxy (–OH) groups of natural purine nucleotides after obtaining the precursor from a modified RNA library involving 2′-fluoro (–F) analogs of uridine and cytidine and natural adenosine and guanosine (Figure 1). The post-SELEX modifications have been successful in rendering nuclease resistance but required considerable time and effort because binding affinities could be markedly decreased or eliminated, depending on the position of the replacement. To overcome this problem, T7 RNA polymerase double-mutant Y639F/H784A was used for enzymatic preparation of the modified RNA library in the SELEX processes, and 2′-OMe RNA aptamers specific to VEGF have been successfully screened directly [6]. One of the 2′-OMe RNA aptamers that could be minimized to 23-mer (which is an unusual short length) was found to be quite stable, and no degradation was observed after incubation at 37°C for 96 h in plasma. Despite being successful for direct screening, structural minimizing, and biostability enhancing, these aptamers were found to have binding affinities in a low nanomolar range that were inferior to those of pegaptanib and its precursors.


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

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

Preparation scheme for chemically modified nucleic acid aptamers that bind to VEGF. High nuclease-resistant 2′-methoxy nucleotides were introduced through Post-SELEX modification process (left), and fully modified 2′-OMe RNA aptamers were directly selected from a library of 2′-OMe transcripts (right).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Preparation scheme for chemically modified nucleic acid aptamers that bind to VEGF. High nuclease-resistant 2′-methoxy nucleotides were introduced through Post-SELEX modification process (left), and fully modified 2′-OMe RNA aptamers were directly selected from a library of 2′-OMe transcripts (right).
Mentions: Owing to the limited tolerance for modified substrates of the RNA polymerase (T7 RNA polymerase) used for SELEX, the 2′-methoxy (–OMe) groups need to be replaced with 2′-hydroxy (–OH) groups of natural purine nucleotides after obtaining the precursor from a modified RNA library involving 2′-fluoro (–F) analogs of uridine and cytidine and natural adenosine and guanosine (Figure 1). The post-SELEX modifications have been successful in rendering nuclease resistance but required considerable time and effort because binding affinities could be markedly decreased or eliminated, depending on the position of the replacement. To overcome this problem, T7 RNA polymerase double-mutant Y639F/H784A was used for enzymatic preparation of the modified RNA library in the SELEX processes, and 2′-OMe RNA aptamers specific to VEGF have been successfully screened directly [6]. One of the 2′-OMe RNA aptamers that could be minimized to 23-mer (which is an unusual short length) was found to be quite stable, and no degradation was observed after incubation at 37°C for 96 h in plasma. Despite being successful for direct screening, structural minimizing, and biostability enhancing, these aptamers were found to have binding affinities in a low nanomolar range that were inferior to those of pegaptanib and its precursors.

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.