Table 9.6.1.
Modified Nucleotides and Examples of Successful In Vitro Aptamer or Ribozyme/Deoxyribozyme Selections, if available.
| MODIFIED NUCLEOTIDE | POLYMERASE | TARGET/FUNCTION (CITATION)α | SPECIAL NOTES |
|---|---|---|---|
| Modified Sugars | |||
| 2’-Amino Pyrimidines* | Y639F T7 RNA Polymerase* |
VEGF/VPF (Green et al., 1995), Human neutrophil elastase (Lin et al., 1994), bFGF (Jellinek et al., 1995), hKGF (Pagratis et al., 1997), IFN-γ (Kubik et al., 1997) Ribozyme – trans cleavage of RNA (Beaudry et al., 2000) |
Several 2'-amino pyrimidine modified RNA aptamers have encountered synthesis difficulties and therefore have been abandoned as therapeutic candidates (reviewed in Keefe and Cload, 2008). |
| 2’-Fluoro Pyrimidines* | Y639F T7 RNA Polymerase* | VEGF 165 (Ruckman et al., 1998 and Chakravarthy et al., 2006), hKGF (Pagratis et al., 1997), IFN-γ (Kubik et al., 1997) | VEGF 165 aptamer is the first human aptamer therapeutic (Macugen ®). The aptamer was post modified after selection to include 2' O-Methyl nucleotides in a process called "back filling". |
| 2’-O-Methyl Nucleotides* | Y693F/H784A (Brieba and Sousa, 2000), "RGVG" (Chelliserrykattil and Ellington, 2004), "2P16" (Siegmund et al., 2012), or R425C T7 RNA Polymerase (Ibach et al., 2013) | VEGF 165 (Burmeister et al., 2005) | This nucleotide is less expensive to synthesize than 2'-amino or 2'-fluoro nucleotides. Also, 2'-O-methyl is a common post-transcriptional modification; for example, there are over 100 2'-O-methyl nucleotides are in each ribosome (Maden, 1990). Thus, the fact that these nucleotides are naturally occurring may make FDA approval more easily attainable. All 23 nucleotides in anti-VEGF aptamer are 2'-O-methyl nucleotides (Burmeister et al., 2005). |
| 2' Compositions, Mixed | Y639F/H784A/K378R T7 RNA Polymerase | Thrombin and IL-23 (Burmeister et al., 2006) | dRmY (dA/dG/mU/mC) was the chosen composition for in vitro selection, although others were considered. |
| TFPI (Waters et al., 2011) | dCmD (mA/mG/mU/dC) was the published composition for in vitro selection, although fRmY (fA/fG/mU/mC) was used in a patented variant against the same target. | ||
| 2’-O,4’-C-Methylene-Bridged ATP, GTP, TTP (Locked Nucleic Acids, LNAs) and 5-Methyl-CTP* | KOD Dash DNA Polymerase* and KOD mutant (KOD 2) DNA Polymerase (Kuwahara et al., 2000) (19:1 ratio of polymerases) (Kasahara et al., 2013) | Thrombin (Kasahara et al., 2013) | KOD Dash DNA polymerase is a mixture of a KOD polymerase and an archaeal DNA polymerase with proofreading activity (available from Toyobo Co. Ltd. (Osaka, Japan)). In the selection, 2’-O,4’-C-methylene bridged (B/L) TTP was incorporated enzymatically, whereas the B/L ATP, B/L GTP, and 5-methyl-CTP were present only in the pool (forward) primer region (Kasahara et al., 2013). |
| 4'-Thio Pyrimidines* | T7 RNA Polymerase* | Thrombin (Kato et al., 2005) | The thio-modified aptamer is 50 times more stable in the presence of RNase A and has an increase in thrombin inhibition compared to the corresponding unmodified RNA aptamer (Kato et al., 2005). |
| Modified Bases | |||
| 5-(3-Aminoally) Deoxycytidine* | Vent (exo -) DNA Polymerase* | RNase DNAzyme (Hollenstein et al., 2013) | This modified DNAzyme contains 3 modified nucleotides (5-guanidinoallyl-dUTP, 5-aminoallyl-dCTP, and 5-imidazolyl-dATP) and cleaves all-RNA targets independent of M2+ (Hollenstein et al., 2012). |
| 5-(3-Aminoallyl) Deoxyuridine* | Sequenase 2.0 DNA Polymerase* | RNase DNAzyme, sequence directed (Perrin et al., 2001) | This RNase DNAzyme contains both the 8-[2-(4-Imidazolyl)ethylamino] deoxyadenosine and the 5-(3-aminoallyl) deoxyuridine modified nucleotides and self-cleaved the internal rC. This is the first example of a metal-independent DNAzyme (Perrin et al., 2001). |
| 5-N-(6-Aminohexyl)Carbamoylmethyl Deoxyuridine | KOD DNA Polymerase* | Thalidomide (Shoji et al., 2007) | This aptamer is highly specific for the R-enantiomer of thalidomide (Shoji et al., 2007). |
| 5-(3-Aminopropynyl) Deoxyuridine | Vent DNA Polymerase* | ATP, ADP, and AMP (Battersby et al., 1999) | This is the first example of the incorporation of a positively charged functional group (Battersby et al., 1999). |
| 5-Benzylaminocarbonyl Deoxyuridine (BndU) (Vaught et al., 2010) | KOD DNA Polymerase* | Plasminogen Activator Inhibitor-1 (PAI-1), as well as multiple other targets (Gold et al., 2010) | Modified nucleotides were used to select against thirteen human proteins ("difficult targets") for which unmodified RNA / DNA in vitro selection did not yield an aptamer. The extent to which a given nucleotide modification was beneficial for a selection was highly dependent on the protein target (Gold et al., 2010). |
| 5-Boronic acid-modified Thymidine | Taq DNA Polymerase* | Fibrinogen (Li et al., 2008) | |
| 5-Bromo Deoxyuridine* | Taq DNA Polymerase* (Brody et al. 1999 and Golden et al. 2000) or E. coli DNA Polymerase I* (Smith et al. 2003) | bFGF (Brody et al. 1999 and Golden et al. 2000) and GP120MN (Smith et al. 2033) | Photocrosslinking aptamers were generated with this modified nucleotide. |
| 5-Carboxamide-modified Deoxyuridine (Vaught et al., 2010) | Deep Vent and KOD XL DNA Polymerases* | Tumor necrosis factor receptor super family member 9 (TNFRSF9) (Vaught et al., 2010) | PCR amplification could not be performed using any of the carboxide derivatives of dUTP, only primer extensions. Therefore, an intermediate PCR step using unmodified nucleotides was required to exponentially amplify, and then subsequent primer extension reactions reincorporated the modified nucleotides. Additionally, amide linkages increase the possibility of hydrogen bonding with the target (Vaught et al., 2010). |
| 5-Guanidinoallyl Deoxyuridine* | Vent (exo -) DNA Polymerase* | RNase DNAzyme (Hollenstein et al., 2013) | See "SPECIAL NOTES" for 4-(3-Aminoallyl) Deoxycytidine. |
| 5-Isobutylaminocarbonyl Deoxyuridine (iBudU) (Vaught et al., 2010) | KOD DNA Polymerase* | Human mobility group -1 (HMG-1), as well as multiple other targets (Gold et al., 2010) | See "SPECIAL NOTES" for 5-Benzylaminocarbonyl Deoxyuridine. |
| 5-Imidazolyl Deoxyadenosine* | Vent (exo -) DNA Polymerase* | RNase DNAzyme (Hollenstein et al., 2013) | See "SPECIAL NOTES" for 4-(3-Aminoallyl) Deoxycytidine. |
| 5-Imidizole Uridine | T7 RNA Polymerase* | Ribozyme with amide synthase activity (Wiegand et al., 1997); Ribozyme with urea synthase activity (Nieuwlandt et al., 2003) | A side-by-side comparison of a 5-imidazol uridine RNA pool versus an unmodified RNA pool used in a selection for urea bond catalysts resulted with significant catalytic activity of the modified pool after nine rounds of selection, while no significant increase in catalytic activity was observed over background with the unmodified RNA selection after fourteens rounds of selection (Nieuwlandt et al., 2003) |
| 5-Imidizole-Uridine analog (unnamed) | Thermostable DNA Polymerases* (possibly Taq, Vent, Pfu, and rTh DNA polymerases) | RNase DNAzyme, sequence directed (Santoro et al., 2000) | |
| 5-Naphtylmethylaminocarbonyl Deoxyuridine (NapdU) (Vaught et al., 2010) | KOD DNA Polymerase* | Human protein targets (Gold et al., 2010) | See "SPECIAL NOTES" for 5-Benzylaminocarbonyl Deoxyuridine. |
| 5-(2-Naphtylmethylaminocarbonyl) Deoxyuridine (2NapdU) (Vaught et al., 2010 and Ochsner et al., 2013) | KOD DNA Polymerase* | C. difficile toxins (Ochsner et al, 2013) | |
| 5-(1-Pentynyl) Deoxyuridine | Vent DNA Polymerase* | Thrombin (Latham et al., 1994) | |
| 5-Phenylethyl Deoxyuridine (PEdU) (Vaught et al., 2010 and Ochsner et al. 2013) | KOD DNA Polymerase* | C. difficile toxins (Ochsner et al, 2013) | |
| 5-Pyridylmethylcarboxamid Uridine | Not noted in paper | Ribozyme with Diels-Alderase activity (Tarasow et al., 1997) | |
| 5-Tyrosyl Deoxyuridine (TyrdU) (Vaught et al., 2010 and Ochsner et al., 2013) | KOD DNA Polymerase* | C. difficile toxins (Ochsner et al, 2013) | |
| 5-Tryptaminocarbonyl Deoxyuridine (TrpdU) (Vaught et al., 2010) | KOD DNA Polymerase* | Fractalkine(CX3CL-1), as well as multiple other targets (Gold et al., 2010) | See "SPECIAL NOTES" for 5-Benzylaminocarbonyl Deoxyuridine. |
| 6-Aminohexyl Adenosine | T7 RNA Polymerase* | Ribozyme with ligase activity (Teramoto et al., 2000) | The ribozyme catalyzed the ligation to its 5' end (Teramoto et al., 2000). |
| 7-(2-Thienyl)Imidazo[4,5-b] Pyridine* | AccuPrime Pfx DNA Polymerase* | VEGF 165 and IFN-γ (Kimoto et al., 2013) | Modified nucleotide exclusively pairs with diol-modified 2-nitro-4-propynlpyrrole, in essence creating a third base pair. The modified aptamers have >100-fold binding affinity over the unmodified aptamers (Kimoto et al., 2013). |
| 8-[2-(4-Imidazolyl)Ethylamino] Deoxyadenosine | Sequenase 2.0 DNA Polymerase* | RNase DNAzyme, sequence directed (Perrin et al., 2001) | See "SPECIAL NOTES" for 5-(3-Aminoallyl) Deoxyuridine. |
| Modified Phosphate Backbone | |||
| Boranophosphate linkages | T7 RNA Polymerase* | ATP (Lato et al., 2002) | |
| Phosphorothioate Linked DNA* (S-linked dNTPs) | Taq DNA Polymerase* |
NF-kB RelA (p65) (King et al., 2002) p50 (King et al., 2002) NF-IL6 (King et al., 1998) |
These modifications are nuclease resistant and efficiently internalized by cells (King et al., 2002). |
| Phosphorothioate Linked RNA* (S-linked NTPs) | T7 RNA Polymerase* | bFGF (Jhaveri et al., 1998) | |
*
Denotes the modified nucleotide or polymerase is commercially available
a
Abbreviations: bFGF, basic fibroblast growth factor; hKGF, human keratinocyte growth factor; IFN-γ, interferon γ; NF-IL6, nuclear factor for human interleukin 6; VEGF, vascular endothelial growth factor; VPF, vascular permeability factor.