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Nucleotides for SELEX/Aptamer Modification

Aptamers are short single-stranded DNA or RNA oligonucleotides (< 100 nt). They gained increasing importance in drug discovery due to their inherent ability to form defined three-dimensional structures that enables them to bind to various targets (e.g. proteins) with antibody-antigen-like affinity and specificity[1].

Aptamers with the highest binding affinity and specificity against a given target molecule are generated by an multi-step in vitro selection and enzymatic amplification process called "systematic evolution of ligands by exponential enrichment (SELEX) (for detailed information please refer to the references below[2,3]).

The main advantages of aptamers compared to antibodies are heat stability, the lack of immunogenicity and minimal interbatch variability. Their usage in diagnostics however, is often hampered by nuclease-mediated degradation.

Enzymatic incorporation of fluoro-, amino- or O-methyl- 2'-ribose-modified nucleotides (1) by generating an initially modified combinatorial library or (2) post-SELEX markedly improve the nuclease resistance of aptamers (Tab. 1) [3-12]. Furthermore, a number of modified nucleotides suitable for the selection of cross-linking capable aptamers (Photo-SELEX) are available as well (Tab. 1)[13-15].

Table 1: SELEX-compatible modified nucleotides.

n/a: not applicable

NucleotideModificationDNA aptamer selection
RNA aptamer selection
Ribose Moiety2'F-dUTPSubstitution of 2'-OH by fluor (F)[5][6,7]
2'OMe-UTPModification of 2'-OH by a methyl group (CH3)
2'NH2-dUTPSubstitution of 2'-OH by an amino group (NH2)


LNA-ATPLNA (Locked Nucleic Acid) with methylene bridge beetween 2'-O and 4'-C


Base Moiety5Br-dUTPModification of C-5 by Bromine (Br)[13]n/a
5I-UTPModification of C-5 by Iodine (I)n/a[14]
4-Thio-UTPSubstitution of 4-O by Sulfur (S)n/a[15]

Selected References

[1] Hermann et al. (2000) Adaptive recognition by nucleic acid aptamers. Science 287:820.
[2] Stoltenburg et al. (2007) SELEX-A (r)evolutionary method to generate high-affinity nucleic acids ligands. Biomolecular Engineering 24:381.
[3] Lauridsen et al. (2012) Enzymatic Recognition of 2'-Modified Ribonucleoside 5'-Triphosphates: Towards the Evolution of Versatile Aptamers. Chem. Bio. Chem. 13:19.
[4] Keefe et al. (2008) SELEX with modified nucleotides. Current Opinion in Chemical Biology 12:448.
[5] Ono et al. (1997) 24-Fluoro modified nucleic acids: polymerase-directed synthesis, properties and stability to analysis by matrix-assisted laser desorption/ionization mass spectrometry. Nucleic Acids Research 25 (22):4581.
[6] Aurup et al. (1992) 2'-Fluoro and 2-amino-2'-deoxynucleoside 5'-triphosphates as substrates for T7 RNA polymerase. Biochemistry 31 (40):9626.
[7] Adler et al. (1998) POST-SELEX Chemical Optimization of a trypanosome-specific RNA aptamer. Comb. Chem. High Throughput Screen. 11 (1):16.
[8] Burmeister et al. (2005) Direct In Vitro Selection of a 2'-O-Methyl Aptamer to VEGF. Chemistry & Biology 12:25.
[9] Burmeister et al. (2006) 2'-Deoxy Purine, 2'-O-Methyl Pyrimidine (dRmY) Aptamers as Candidate Therapeutics. OLIGONUCLEOTIDES 16:337.
[10] Padilla et al. (1999) Efficient synthesis of nucleic acids heavily modified with non-canonical ribose 2'-groups using a mutant T7 RNA Polymerase (RNAP). Nucleic Acids Res. 27 (6):156.
[11] Padilla et al. (2002) A Y639F/H784A T7 RNA polymerase double mutant displays superior properties for synthesizing RNAs with non-canonical NTPs. Nucleic Acids Res. 30 (24):e138.
[12] Lin et al. (1994) Modified RNA sequence pools for in vitro selection. Nucleic Acids Res. 22 (24):5229.
[13] Golden et al. (2000) Diagnostic potential of PhotoSELEX-evolved ssDNA aptamers. Journal of Biotechnology 81:167.
[14] Jensen et al. (1995) Using in vitro selection to direct the covalent attachment of human immunodeficiency virus type 1 Rev protein to high-affinity RNA ligands. Proc. Natl. Acad. Sci. USA 92:12220.
[15] Park et al. (2008) Higher-Order Association States of Cellular ERBB3 Probed with Photo-Cross-Linkable Aptamers. Biochemistry 47 (46):11992.
[16] Crouzier et al. (2012) Efficient reverse transcription using locked nucleic acid nucleotides towards the evolution of nuclease resistant RNA aptamers. PLoS One. 7 (4):e35990.