Nucleotides for Drug Discovery

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SELEX/Aptamer Modification

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

Nucleotide Modification DNA aptamer selection
(DNA SELEX)		 RNA aptamer selection
(RNA SELEX)
Ribose Moiety 2’F-dUTP Substitution of 2′-OH by fluor (F) [5] [6,7]
2’F-dCTP [5] [6,7]
2’F-dATP [5] [6]
2’F-dGTP [5]
2’OMe-UTP Modification of 2′-OH by a methyl group (CH3) [8,9,10,11]
2’OMe-CTP [8,9,11]
2’OMe-ATP [8]
2’OMe-GTP [8]
2’NH2-dUTP Substitution of 2′-OH by an amino group (NH2) [6,12]
2’NH2-dCTP [12]
2’NH2-dATP
2’NH2-dGTP
LNA-ATP LNA (Locked Nucleic Acid) with methylene bridge beetween 2′-O and 4′-C [16]
LNA-GTP
LNA-CTP
LNA-UTP
Base Moiety 5Br-dUTP Modification of C-5 by Bromine (Br) [13] n/a
5I-UTP Modification of C-5 by Iodine (I) n/a [14]
4-Thio-UTP Substitution of 4-O by Sulfur (S) n/a [15]

 

 

Name Cat. No. Size
2′-Fluoro-dUTP NU-1215S 50 μl (100 mM)
2′-Fluoro-dUTP NU-1215L 5 x 50 μl (100 mM)
2′-Fluoro-dCTP NU-1214S 50 μl (100 mM)
2′-Fluoro-dCTP NU-1214L 5 x 50 μl (100 mM)
2′-Fluoro-dATP NU-151S 50 μl (100 mM)
2′-Fluoro-dATP NU-151L 5 x 50 μl (100 mM)
2′-Fluoro-dGTP NU-1216S 50 μl (100 mM)
2′-Fluoro-dGTP NU-1216L 5 x 50 μl (100 mM)
2′-Fluoro-dNTP Bundle NU-10405-20 4 x 50 μl (4 x 5 μmol)
2’OMe-UTP NU-1212S 50 μl (100 mM)
2’OMe-UTP NU-1212L 5 x 50 μl (100 mM)
2’OMe-CTP NU-1211S 50 μl (100 mM)
2’OMe-CTP NU-1211L 5 x 50 μl (100 mM)
2’OMe-ATP NU-1184S 50 μl (100 mM)
2’OMe-ATP NU-1184L 5 x 50 μl (100 mM)
2’OMe-GTP NU-1127S 50 μl (100 mM)
2’OMe-GTP NU-1127L 5 x 50 μl (100 mM)
2’NH2-dUTP NU-242S 50 μl (100 mM)
2’NH2-dUTP NU-242L 5 x 50 μl (100 mM)
2’NH2-dCTP NU-243S 50 μl (100 mM)
2’NH2-dCTP NU-243L 5 x 50 μl (100 mM)
2’NH2-dATP NU-244S 50 μl (100 mM)
2’NH2-dATP NU-244L 5 x 50 μl (100 mM)
2’NH2-dGTP NU-245S 50 μl (100 mM)
2’NH2-dGTP NU-245L 5 x 50 μl (100 mM)
LNA-ATP NU-982 10 μl (100 mM)
LNA-GTP NU-983 10 μl (100 mM)
LNA-CTP NU-984 10 μl (100 mM)
LNA-UTP NU-985 10 μl (100 mM)
5-Bromo-dUTP NU-122S 50 μl (10 mM)
5-Bromo-dUTP NU-122L 5 x 50 μl (10 mM)
5-Iodo-UTP NU-119S 10 μl (100 mM)
5-Iodo-UTP NU-119L 5 x 10 μl (100 mM)
4-Thio-UTP NU-1156S 10 μl (100 mM)
4-Thio-UTP NU-1156L 5 x 10 μl (100 mM)

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.

mRNA Modification

Nucleotides for mRNA Modification

Synthetic messenger RNA (mRNA) can be used to deliver exogenous genetic information inside cells that is converted by the cellular translational machinery into the encoded protein which subsequently induces a cellular response depending on its biological function.

Therapeutic protein encoding mRNAs have most recently gained increasing attention as versatile protein delivery molecules in non-viral gene therapy mostly due to their superior safety profile compared to plasmid DNA-based approaches (inability to integrate into the host genome)[1]. In addition, high-purity mRNA is easily synthesized even large scales by phage RNA polymerase-mediated in vitro transcription[2,3].

Their application however, was so far been hampered by the limited stability and strong immunogenicity of in vitro transcribed mRNAs subsequently leading to increased expression rates of the respective protein.

Enzymatic incorporation of a set several nucleotide analogs (single or combined) during the transcription process markedly improves the pharmacokinetic properties of in vitro transcribed mRNA both in terms of stability (increased nuclease resistance) and decreased immunogenicity (Fig. 1)[4-8].

Find more Cap analogs in the 5′-Capping section! Kits for enzymatic RNA Synthesis and poly(A) tailing are available as well.

ARCA-, 5-methylcytidine- & pseudouridine-based mRNA Synthesis Kits

Figure 1: Pharmacokinetic properties of synthetic mRNA are improved by nucleotide analog incorporation during in vitro RNA synthesis.
ARCA: “anti-reverse” cap analog, m5CTP = 5′-methyl-cytidine triphosphate, m6ATP=N6-methyl-adenosine-5′-triphosphate, s2UTP = 2-thio-uridine triphosphate, Ѱ = pseudouridine triphosphate, me1Ѱ = N1Methylpseudouridine triphosphate, 5moUTP = 5-Methoxyuridine triphosphate.

 

Name Cat. No. Size
m27,3′-OGP3G (ARCA Cap Analog) – Solid NU-855-1 1 mg
m27,3′-OGP3G (ARCA Cap Analog) – Solid NU-855-1 1 mg
m27,3′-OGP3G (ARCA Cap Analog) – Solid NU-855-5 5 mg
m27,3′-OGP3G (ARCA Cap Analog) – Solution NU-855S 10 μl (100 mM)
m27,3′-OGP3G (ARCA Cap Analog) – Solution NU-855L 5 x 10 μl (100 mM)
5-Methyl-CTP NU-1138S 10 μl (100 mM)
5-Methyl-CTP NU-1138L 5 x 10 μl (100 mM)
N6-Methyl-ATP NU-1101S 10 μl (100 mM)
N6-Methyl-ATP NU-1101L 5 x 10 μl (100 mM)
2-Thio-UTP NU-1151S 10 μl (100 mM)
2-Thio-UTP NU-1151L 5 x 10 μl (100 mM)
Pseudo-UTP NU-1139S 10 μl (100 mM)
Pseudo-UTP NU-1139L 5 x 10 μl (100 mM)
N1-Methylpseudo-UTP NU-890S 10 μl (100 mM)
N1-Methylpseudo-UTP NU-890L 5 x 10 μl (100 mM)
5-Methoxy-UTP NU-972S 10 μl (100 mM)
5-Methoxy-UTP NU-972L 5 x 10 μl (100 mM)
N1-Methyl-ATP – Solid NU-1027-1 1 mg
N1-Methyl-ATP – Solid NU-1027-5 5 mg
N1-Methyl-ATP – Solution NU-987S 10 μl (100 mM)
N1-Methyl-ATP – Solution NU-987L 5 x 10 μl (100 mM)
N4-Acetyl-CTP NU-988S 10 μl (100 mM)
N4-Acetyl-CTP NU-988L 5 x 10 μl (100 mM)

Selected References

[1] Tavernier et al. (2011) mRNA as gene therapeutic: How to control protein expression. Journal of controlled disease 150:238.
[2] Karikó et al. (2011) Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA. Nuclei Acids Res. 39 (21):9329.
[3] Pascolo et al. (2006) Vaccination with messenger RNA. In: Methods in Molecular Medicine 127 (Saltzmann). Humana Press.
[4] Kormann et al. (2011) Expression of therapeutic proteins after delivery of chemically modified mRNA in mice. Nature Biotechnology 29 (2):154.
[5] Karikó et al. (2008) Incorporation of Pseudouridine into mRNA Yields Superior Nonimmunogenic Vector With Increased Translational Capacity and Biological Stability. Mol. Ther. 16 (11):1833.
[6] Karikó et al. (2005) Suppression of RNA Recognition by Toll-like Receptors: The Impact of Nucleoside Modification and the Evolutionary Origin of RNA. Immunity 23:165.
[7] Anderson et al. (2011) Nucleoside modifications in RNA limit activation of 2′-5′-oligoadenylate synthetase and increase resistance to cleavage by RNAse L. Nuclei Acids Res. 39 (21):9329.
[8] Warren et al. (2011) Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mRNA. Cell Stem Cell 7:618.
[9] Andies et al. (2015) N (1)-methylpseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice. J. Control. Release 217:337.
[10] Li et al. (2016) Effects of Chemically Modified Messenger RNA on Protein Expression Bioconjugate Chem. 27 (3):849.

Antiviral Nucleotides

Antiviral Nucleotides

Antiviral nucleotides are nucleotide analogs that repress the viral reproduction by interfering with several key mechanisms of viral nucleic acid replication. They compete with natural dNTP/NTP substrates for the incorporation into the nascent viral nucleic acid thereby leading to chain termination or mutagenesis (Tab. 2).

In vivo or cell culture experiments can be performed using the non-phosphorylated nucleoside variant of an antiviral nucleotide that is intracellularly metabolized by multiple viral and/or host kinases to its active, the antiviral effect exerting (tri)phosphorylated forms (NMP → NDP → NTP)[7,8].

Alternatively, a number of mono- and triphosphorylated forms of antiviral nucleotides are available for functional in vitro experiments (Tab. 1).

Please refer to the corresponding data sheet for detailed application data.

Table 1: Available antiviral nucleoside and nucleotide analogs.

NucleosideNucleotide Monophosphate (NMP)Nucleotide Triphosphate (NTP)
3TC (Lamivudine)3TCMP3TCTP
d4T (Stavudine)d4TMPd4TTP
AzT (Zidovudine)AzTMPAzTTP
ara-A (Vidarabine)ara-AMPara-ATP
AciclovirAciclovir monophosphateAciclovir triphosphate
RibavirinRibavirin monophosphateRibavirin triphosphate
TelbivudineTelbivudine monophosphateTelbivudine triphosphate
6-Aza-U6-Aza-UMP6-Aza-UTP
TenofovirTenofovir diphosphate

Table 2: Antiviral properties of selected nucleotide analogs.

NucleotideAnalog of…Incorporation by viral…Mechanism of inhibition
Lamivudine triphosphate…Cytidine triphosphate…Reverse Transcriptase[1]Chain termination of viral cDNA synthesis
Stavudine triphosphate…Thymidine triphosphate…Reverse Transcriptase[2]Chain termination of viral cDNA synthesis
Zidoduvine triphosphate…Thymidine triphosphate…Reverse Transcriptase[3]Chain termination of viral cDNA synthesis
Aciclovir triphosphate…Guanosine triphosphate…DNA polymerase[4]Chain termination of viral DNA synthesis[4]
Vidarabine triphosphate…Adenosine triphosphate…DNA polymerase[5]unknown
Ribavirin triphosphate…Guanosine triphosphate…RNA polymerase[6]Mutagenesis of viral genome replication[6]
Name Cat. No. Size
3TCMP NU-1605S 10 μl (10 mM)
3TCMP NU-1605L 5 x 10 μl (10 mM)
3TCTP NU-1606S 5 μl (10 mM)
3TCTP NU-1606L 5 x 5 μl (10 mM)
d4TMP NU-1603S 20 μl (10 mM)
d4TMP NU-1603L 5 x 20 μl (10 mM)
d4TTP NU-1604S 10 μl (100 mM)
d4TTP NU-1604L 5 x 10 μl (100 mM)
AzTMP NU-1601S 20 μl (10 mM)
AzTMP NU-1601L 5 x 20 μl (10 mM)
AzTTP NU-989S 10 μl (100 mM)
AzTTP NU-989L 5 x 10 μl (100 mM)
ara-Adenosine-5′-monophosphate (ara-AMP) NU-875 2 mg
ara-Adenosine-5′-triphosphate (ara-ATP) NU-1111S 50 μl (10 mM)
ara-Adenosine-5′-triphosphate (ara-ATP) NU-1111L 5 x 50 μl (10 mM)
Acyclovir-5′-monophosphate NU-876 2 mg
Acyclovir-5′-triphosphate NU-877 2 mg
Ribavirin-5′-monophosphate NU-021-2 2 mg
Ribavirin-triphosphate NU-1105S 20 μl (10 mM)
Ribavirin-triphosphate NU-1105L 5 x 20 μl (10 mM)
6-Aza-UTP NU-896S 10 μl (100 mM)
6-Aza-UTP NU-896L 5 x 10 μl (100 mM)
Tenofovir NU-974 100 mg
Tenofovir-diphosphate NU-975 1 mg
AzTTP NU-989S 10 μl (100 mM)
AzTTP NU-989L 5 x 10 μl (100 mM)
Ganciclovir-triphosphate NU-275S 10 μl (100 mM)
Ganciclovir-triphosphate NU-275L 5 x 10 μl (100 mM)
Adefovir NU-281 100 mg
Ganciclovir-triphosphate NU-275S 10 μl (100 mM)
Ganciclovir-triphosphate NU-275L 5 x 10 μl (100 mM)

Selected References

1] Huang at al. (2011) Effect of reverse transcriptase inhibitors on Line-1 and Ty1 reverse transcriptase activities and on LINE-1 retrotransposition. BMC Biochemistry 12:18.
[2] Vaccaro at al. (1999) Mechanism of Inhibition of the Human Immunodeficiency Virus Type 1 Reverse Transcriptase by d4TTP: an Equivalent Incorporation Efficiency Relative to the Natural Substrate dTTP. Antimicrob. Agents Chemother. 44 (1):217.
[3] Jaju at al. (1995) Human immunodeficiency virus type 1 reverse transcriptase. 3′-Azidodeoxythymidine 5′-triphosphate inhibition indicates two-step binding for template-primer. J. Biol. Chem. 270 (17):9740.
[4] Furmann at al. (1979) Inhibition of Herpes Simplex Virus-Induced DNA Polymerase Activity and Viral DNA Replication by 9-(2-Hydroxyethoxymethyl)guanine and Its Triphosphate. J. Virol. 29 (2):154.
[5] Muller at al. (1977) Inhibition of Herpesvirus DNA synthesis by 9-O-D-Arabinofuranosyladenine in cellular and cell-free systems. Ann. N.Y. Acad. Sci. 284:34.
[6] Crotty at al. (2000) The broad-spectrum antiviral ribonucleoside ribavirin is an RNA virus mutagen. Nature Medicine 6 (12):1375.
[7] Ray at al. (2009) Metabolism of antiviral nucleosides and nucleotides. In: Antiviral Research: Strategies in antiviral drug discovery (LaFemina). ASM Press.
[8] Simons at al. (2005) Recent Advances in Antiviral Nucleoside and Nucleotide Therapeutics. Current Topics in Medicinal Chemistry 5:1191.

Cytostatic Nucleotides

Cytostatic Nucleotides

A hallmark of cancer is uncontrolled cell proliferation caused by deregulation of multiple signaling pathways associated with cell replication, metabolism and programmed cell death[1].

Cytostatic nucleotides are substrate analogs of physiological nucleotides that inhibit cell proliferation by interfering with cellular targets involved in DNA/RNA synthesis and nucleotide metabolism.

In vivo or cell culture experiments can be performed using the non-phosphorylated nucleoside variant of a cytostatic nucleotide that is metabolized by multiple cellular kinases to its active phosphorylated forms (NMP → NDP → NTP).

Alternatively, a number of mono-, di- and triphosphorylated analogs of cytostatic nucleotides are available for functional in vitro experiments (Tab. 1).

Please refer to the corresponding data sheet for detailed application data.

Table 1: Available cytostatic nucleosides and corresponding nucleotide variants.

Nucleotide Monophosphate (NMP)Nucleotide Diphosphate (NDP)Nucleotide Triphosphate (NTP)
ara-A (Vidarabine)ara-AMPara-ATP
Cl-F-ara-A (Clofarabine)Cl-F-ara-AMPCl-F-ara-ATP
2’Cl-Adenosine2’Cl-dATP
ara-C (Cytarabine)ara-CMPara-CDPara-CTP
dF-dC (Gemcitabine)dF-dCMPdF-dCDPdF-dCTP
5F-U (5-Fluoruracil)5F-dUMP5F-dUTP
6-Thio-I (6-Thio-inosine)6-Thio-IMP6-Thio-IDP6-Thio-ITP
6-Methylthio-I (6-Methylthio-inosine)6-Methylthio-IMP6-Methylthio-IDP6-Methylthio-ITP
5-Aza-dC (Decitabine)5-Aza-dCTP
Name Cat. No. Size
ara-Adenosine-5′-monophosphate (ara-AMP) NU-875 2 mg
ara-Adenosine-5′-triphosphate (ara-ATP) NU-1111S 50 μl (10 mM)
ara-Adenosine-5′-triphosphate (ara-ATP) NU-1111L 5 x 50 μl (10 mM)
Clofarabine-5′-monophosphate NU-873 3 mg
Clofarabine-5′-triphosphate NU-874 5 μmol
2′-Chloro-dATP NU-148S 50 μl (10 mM)
2′-Chloro-dATP NU-148L 5 x 50 μl (10 mM)
ara-Cytidine-5′-monophosphate (ara-CMP) NU-872 2 mg
ara-Cytidine-5′-diphosphate (ara-CDP) NU-1171S 50 μl (10 mM)
ara-Cytidine-5′-diphosphate (ara-CDP) NU-1171L 5 x 50 μl (10 mM)
ara-Cytidine-5′-triphosphate (ara-CTP) NU-1170S 100 μl (10 mM)
ara-Cytidine-5′-triphosphate (ara-CTP) NU-1170L 5 x 100 μl (10 mM)
Gemcitabine-5′-monophosphate NU-871 2 mg
Gemcitabine-5′-diphosphate NU-1608S 20 μl (10 mM)
Gemcitabine-5′-diphosphate NU-1608L 5 x 20 μl (10 mM)
Gemcitabine-5′-triphosphate NU-1607S 50 μl (10 mM)
Gemcitabine-5′-triphosphate NU-1607L 5 x 50 μl (10 mM)
5-Fluoro-dUMP NU-153S 50 μl (10 mM)
5-Fluoro-dUMP NU-153L 5 x 50 μl (10 mM)
5-Fluoro-dUTP NU-154S 20 μl (100 mM)
5-Fluoro-dUTP NU-154L 5 x 20 μl (100 mM)
6-Mercaptopurine-riboside-5′-monophosphate NU-1148S 100 μl (10 mM)
6-Mercaptopurine-riboside-5′-monophosphate NU-1148L 5 x 100 μl (10 mM)
6-Mercaptopurine-riboside-5′-diphosphate NU-1210S 100 μl (10 mM)
6-Mercaptopurine-riboside-5′-diphosphate NU-1210L 5 x 100 μl (10 mM)
6-Mercaptopurine-riboside-5′-triphosphate NU-1110S 100 μl (10 mM)
6-Mercaptopurine-riboside-5′-triphosphate NU-1110L 5 x 100 μl (10 mM)
6-Methylthio-IMP NU-1131S 150 μl (10 mM)
6-Methylthio-IMP NU-1131L 5 x 150 μl (10 mM)
6-Methylthio-IDP NU-1132S 100 μl (10 mM)
6-Methylthio-IDP NU-1132L 5 x 100 μl (10 mM)
6-Methylthio-ITP NU-1133S 150 μl (10 mM)
6-Methylthio-ITP NU-1133L 5 x 150 μl (10 mM)
5-Aza-dCTP NU-1118 20 mg

Selected References

[1] Hanahan et al. (2011) Hallmarks of Cancer: The next generation. Cell 144 (5):646.

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