Bases and modifications for custom synthesis

1-Methyl-guanosine

Modification Code:
m1G

Unit Structure:

Unit Molecular Weight:
359.23

References:
Agris, P. F. et al. Site-selective introduction of modified purine and pyrimidine ribonucleosides into RNA by automated phosphoramidite chemistry. Biochimie 77, 125-134 (1995).

2,6-Diaminopurine

Modification Code:
DAP

Description:
2,6-Diaminopurine (2-aminoadenine, DAP) is an adenine analog which can be found in S-2L cyanophage DNA where 2,6-diaminopurine is completely substituted for adenine. Studies have shown that DAP in DNA is compatible with normal DNA function. The DAP-T base pair possesses an extra hydrogen bond compared with A-T because of the additional -NH₂ pointing toward the minor groove of DNA. DAP is a common tool in nucleic acid chemistry which can be used to study molecular recognition between DNA or RNA and ligands, both small and large. Double-stranded siRNAs having DAP modification incorporated into the antisense strand were capable of inducing RNAi activity.

Unit Structure:

Unit Molecular Weight:
344.22

References:
Cheong, C. et al. Thermodynamic studies of base pairing involcing 2,6-diaminopurine. Nucleic Acids Res. 16, 5115-5122 (1988).
Bailey, C and Waring, M. J. The use of diaminopurine to investigate structural properties of nucleic acids and molecular recognition between ligands and DNA. Nucleic Acids Res. 26, 4309-4314 (1998).
Chiu, Y. and Rana, T. M. siRNA function in RNAi: a chemical modification analysis. RNA 9, 1034-1048 (2003).

2-Methyl-adenosine

Modification Code:
m2A

Unit Structure:

Unit Molecular Weight:
343.24

References:
Agris, P. F. et al. Site-selective introduction of modified purine and pyrimidine ribonucleosides into RNA by automated phosphoramidite chemistry. Biochimie 77, 125-134 (1995).

2-Aminopurine

Modification Code:
2AP

Description:
2-Aminopurine (2AP) is a fluorescent analog of guanosine and adenosine. Because 2AP can base pair with uridine, it can replace normal A:U pairs without substantial deformation of duplexes. 2-Aminopurine is widely used in biochemical settings as a site-specific fluorescent probe of DNA and RNA structure and base-flipping and -folding.

Unit Structure:

Unit Molecular Weight:
329.21

References:
Hall, K. B. 2-Aminopurine as a probe of RNA conformational transition. Methods Enzymol. 469, 269-285 (2009).

4-Thio-uridine

Modification Code:
4-S-U

Description:
4-Thio-uridine (s4U) is a thiol-modified ribonucleoside, and is typically used to modify oligos slated for RNA, or RNA-protein, structural studies. Because 4-thio-rU is photoreactive, 4-thio-rU modified RNA oligonucleotides can be used as photoaffinity probes in the role of substrate analogs for characterizing the enzyme:substrate complex. In addition, because the thiol group is chemically reactive, other moieties can be conjugated at the thiol group of 4-thio-rU. Such a strategy was used to introduce spin labels to 4-thio-rU-containing RNA oligonucleotides.

Unit Structure:

Unit Molecular Weight:
322.23

References:
Kumar R. K. and Davis, D. R. Synthesis and studies on the effect of 2-thiouridine and 4-thiouridine on sugar conformation and RNA duplex stability. Nucleic Acids Res. 25, 1272-1280 (1997).
Yu, Y. T. Site-specific 4-thiouridine incorporation into RNA molecules. Methods Enzymol. 318, 71-88 (2000).
Rublack, N. et al. Synthesis of specifically modified oligonucleotides for application in structural and functional analysis of RNA. J. Nucleic Acids 1-19 (2011).

5-Bromo-uridine

Modification Code:
U[5Br]

Description:
Halogenated nucleosides are used to crosslink oligonucleotides to DNA, RNA, and proteins. They are also used to investigate structures via x-ray diffraction and NMR. Modifications such as 5-bromo-uridine that stabilize base-pairing interactions can be used in designing siRNAs for various applications.

Unit Structure:

Unit Molecular Weight:
385.06

References:
Zeng, Y. and Wang, Y. Sequence-dependent formation of intrastrand crosslink products from the UVB irradiation of duplex DNA containing a 5-bromo-2'-deoxyuridine or 5-bromo-2'-deoxycytidine. Nucleic Acids Res. 34, 6521-6529 (2006).
Javed, A. et al. In situ immunofluorescence analysis: analyzing RNA synthesis by 5-bromouridine-5'-triphosphate labeling. Methods Mol. Biol. 285, 29-31 (2004).
Chiu, Y. and Rana, T. M. siRNA function in RNAi: a chemical modification analysis. RNA 9, 1034-1048 (2003).

5-Fluoro-cytidine

Modification Code:
C[5F]

Description:
Halogenated nucleosides are used to crosslink oligonucleotides to DNA, RNA, and proteins. They are also used to investigate structures via x-ray diffraction and NMR.

Unit Structure:

Unit Molecular Weight:
323.17

References:
Puffer, B. et al. 5-Fluoro pyrimidines: labels to probe DNA and RNA secondary structures by 1D 19F NMR spectroscopy. Nucleic Acids Res. 37, 7728-7740 (2009).

5-Fluoro-uridine

Modification Code:
U[5F]

Description:
Halogenated nucleosides are used to crosslink oligonucleotides to DNA, RNA, and proteins. They are also used to investigate structures via x-ray diffraction and NMR.

Unit Structure:

Unit Molecular Weight:
324.16

References:
Ferrer-Orta, C. et al. Sequential structures provide insight into the fidelity of RNA replication. Proc. Natl. Acad. Sci. USA 104, 9463-9468 (2007).
Puffer, B. et al. 5-Fluoro pyrimidines: labels to probe DNA and RNA secondary structures by 1D ¹⁹F NMR spectroscopy. Nucleic Acids Res. 37, 7728-7740 (2009).

5-Iodo-uridine

Modification Code:
U[5I]

Description:
Halogenated nucleosides are used to crosslink oligonucleotides to DNA, RNA, and proteins. They are also used to investigate structures via x-ray diffraction and NMR. Modifications such as 5-iodo-uridine that stabilize base-pairing interactions can be used in designing siRNAs for various applications.

Unit Structure:

Unit Molecular Weight:
432.06

References:
Stump, W. T. and Hall, K. B. Crosslinking of an iodo-uridine-RNA hairpin to a single site on the human U1A N-terminal RNA binding domain. RNA 1, 55-63 (1995).
Chiu, Y. and Rana, T. M. siRNA function in RNAi: a chemical modification analysis. RNA 9, 1034-1048 (2003).

5-Methyl-cytidine

Modification Code:
5-M-C

Description:
The nucleobase modification 5-methylcytosine (5-mC) is widespread both in DNA and different cellular RNAs. In tRNA, 5-mC sites are typically located around the variable region and anticodon loop and research has shown that tRNA methylation stabilizes tRNA secondary structure, affects aminoacylation and codon recognition, and confers metabolic stability. Additionally, ribosomal RNA methylation was shown to be involved in translational fidelity and tRNA recognition. 5-Methyl-cytosine enhances DNA or RNA duplex stability by elevating melting temperature 0.5 - 1.3°C per addition.

Unit Structure:

Unit Molecular Weight:
319.21

References:
Motorin, Y. et al. 5-Methylcytosine in RNA: detection, enzymatic formation and biological functions. Nucleic Acids Res. 38, 1415-1430 (2010).
Squires, J. E. et al. Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA. Nucleic Acids Res. 40, 5023-5033 (2012).

5-Methyl-deoxycytidine

Modification Code:
5-M-dC

Description:
The nucleobase modification 5-methylcytosine (5-mC) is widespread both in DNA and different cellular RNAs. 5-Methyl-cytosine enhances DNA or RNA duplex stability by elevating melting temperature 0.5 - 1.3°C per addition.

Unit Structure:

Unit Molecular Weight:
303.21

References:

Freier, S. M. and Altmann, K.-H. The ups and downs of nucleic acid duplex stability: structure-stability studies on chemically modified DNA:RNA duplexes. Nucleic Acid Res. 25, 4429-4443 (1997).

5-Methyl-uridine

Modification Code:
rT

Description:
5-Methyluridine, also called ribothymidine, is the ribonucleoside counterpart to the deoxythymidine. Substitution of 5-methyluridine in siRNA guide strand results in gene silencing activities comparable to or better than that of wide-type siRNA. Such major groove modifications also increase the serum stability of siRNAs.

Unit Structure:

Unit Molecular Weight:
335.23

References:
Motorin, Y. and Helm, M. RNA nucleotide methylation. Wiley Interdiscip. Rev. RNA 2, 611-631 (2011).
Terrazas, M. and Kool, E. T. RNA major groove modifications improve siRNA stability and biological activity. Nucleic Acid Res. 37, 346-353 (2009).

Inosine

Modification Code:
I

Description:
The most commonly used degenerate modified base is inosine, which serves as a more-or-less universal base, as it is capable of pairing with all four natural nucleotides, though not with equal affinity (I-C >I-A>I-T~I-G>I-I). Even so, inosine continues to be successfully used in this role in a variety of applications requiring degeneracy at certain base positions of primers and probes, particularly at wobble positions, where degeneracy might be needed to permit annealing to many different, but closely related, sequences.

Unit Structure:

Unit Molecular Weight:
330.19

References:
Loakes, D. The applications of universal DNA base analogues. Nucleic Acids Res. 29, 2437-2447 (2001). Alseth, I. et al. Inosine in DNA and RNA. Cur. Opi. Genetics and Dev. 26, 116-123 (2014).

N3-Methyl-uridine

Modification Code:
3-M-U

Unit Structure:

Unit Molecular Weight:
320.19

References:
Micura, R. et al. Methylation of the nucleobases in RNA oligonucleotides mediates duplex-hairpin conversion. Nucleic Acids Res. 29, 3997-4005 (2001).
Chui, H. M. P. et al. Synthesis of a 3-methyluridine phosphoramidite to investigate the role of methylation in a ribosomal RNA hairpin. Bioorg. Med. Chem. 10, 325-332 (2002).

N6,N6-Dimethyl-adenosine

Modification Code:
DMA

Description:
N6,N6-Dimethyl-adenosine (m⁶₂A) is a minor RNA modification found primarily in rRNA, and recently, in Mycobacterium tRNA. N6-dimethyl rA appears to play a structural role in rRNA.

Unit Structure:

Unit Molecular Weight:
357.26

References:
Politz, S. M. and Glitz, D. G. Ribosome structure: localization of N6,N6-dimethyladenosine by electron microscopy of a ribosome-antibody complex. Proc. Natl. Acad. Sci. USA 74, 1468-1472 (1997).
Rife, J. P. et al. The synthesis of RNA containing the modified nucleotides N2-methylguanosine and N6,N6-dimethyladenosine. Nucleosides and Nucleotides 17, 2281-2288 (1998).
Micura, R. et al. Methylation of the nucleobases in RNA oligonucleotides mediates duplex-hairpin conversion. Nucleic Acids Res. 29, 3997-4005 (2001).

N6-Methyl-adenosine

Modification Code:
m6A

Description:
N6-methyl-adenosine (m⁶A) is the most abundant modification in mammalian mRNA and long non-coding RNA. First discovered in the 1970s, m⁶A modification has been proposed to function in mRNA splicing, export, stability, and immune tolerance.

Unit Structure:

Unit Molecular Weight:
343.24

References:
Pan, T. N6-Methyl-adenosine modification in messenger and long non-coding RNA. Trends Biochem. Sci. 38, 204-209 (2013).

Pseudouridine

Modification Code:
~U

Description:
Pseudouridine (ψ) is one of the most abundant post-transcriptionally modified nucleotides in various stable RNAs of all organisms. Pseudouridine is derived from uridine via base-specific isomerization, resulting in an extra hydrogen-bond donor that distinguishes it from other nucleotides. Pseudouridine-modified RNA can be used as research tools for studies into the roles of this residue in RNA structure and function in the cell.

Unit Structure:

Unit Molecular Weight:
306.17

References:
Sipa, K. et al. Effect of base modifications on structure, thermodynamic stability, and gene silencing activity of short interfering RNA. RNA 13, 1301-1316 (2007).
Ge, J and Yu, Y. T. RNA pesudouridylation: new insights into an old modification. Trends Biochem. Sci. 38, 210-208 (2013).

Purine ribonucleoside

Modification Code:
Pu

Description:
Purine ribonucleoside (Nebularine) lacks exocyclic functional groups and offers an altered hydrogen bonding scheme while retaining base stacking ability. It can be viewed as an adenosine analog with the hydrogen bond donor deleted.

Unit Structure:

Unit Molecular Weight:
314.19

References:
Lucia, J. S. et al. Functional group substitutions as probes of hydrogen bonding between GA mismatches in RNA internal loops. J. Am. Chem. Soc. 113, 4313-4322 (1991).

Pyrrolo-cytidine

Modification Code:
pC

Description:
Pyrrolo-cytidine is a fluorescent bicyclic analog of cytidine that base pairs specifically with guanosine (but not A, C, or U). Its excitation and emission maxima are 350 nm and 450 nm, respectively. Because these values are significantly different from the UV absorption maxima of DNA/RNA (260 nm) and protein (280 nm), pyrrolo-C is useful for probing the structures of DNA/RNA or protein-nucleic acid complexes.

Unit Structure:

Unit Molecular Weight:
343.23

References:
Berry, D. A. et al. Pyrrolo-dC and pyrrolo-C: fluorescent analogs of cytidine and 2'-deoxycytidine for the study of oligonucleotides. Tetrahedron Lett. 45, 2457-2461 (2004).
Tinsley, R. A. and Walter, N. G. Pyrrolo-C as a fluorescent probe for monitoring RNA secondary structure formation. RNA 12, 522-529 (2006).

Ribavirin

Modification Code:
RBV

Description:
Ribavirin is a guanosine ribonucleoside analog used to stop viral RNA synthesis and viral mRNA capping. It displays broad-spectrum anti-viral activity and is currently used for the treatment of some viral infections.

Unit Structure:

Unit Molecular Weight:
306.17

References:
Cameron C. E. and Castro C. The mechanism of action of ribavirin: lethal mutagenesis of RNA virus genomes mediated by the viral RNA-dependent RNA polymerase. Curr. Opin. Infect. Dis. 14, 757-764 (2001).

2'-Amino-butyryl-pyrene-uridine

Modification Code:
2'-P-U

Description:
Pyrene is a polycyclic aromatic hydrocarbon (PAH) consisting of four fused benzene rings. Because pyrene can be chemically modified in various ways and its fluorescence quantum efficiencies are high in both monomers and excimer emissions, pyrene-modified olignucleotides have been exploited as fluorescent probes of DNA and RNA in hybridization assays. In addition, pyrene fluorescence is largely affected by environmental factors, such as solvent and nearby nucleotide bases, which has led to its development as a potential probe for nucleic acid structures.

Unit Structure:

Unit Molecular Weight:
575.51

Pyrene Extinction Coefficient:
54,000

Excitation/Emission Max:
335 nm/381 nm

References:
Yamana, K. et al. 2'-Pyrene modified oligonucleotide provides a highly sensitive probe of RNA. Nucleic Acids Res. 27, 2387-2392 (1999).
Du, H. et al. PhotochemCAD: A computer-aided design and research tools in photochemistry. Photochem. Photobiol. 68, 141-142 (1998).

2'-Amino-cytidine

Modification Code:
2'-N-C

Description:
The thermal stability of duplexes containing 2'-amino-RNA has been determined and it was reported that 2'-amino-C substitutions destabilized by about 4° relative to RNA C. It was also further reported that 2'-amino-RNA linkages are nuclease-resistant. The pKa of the 2'-amino group is quite low at 6.2 but this retains sufficient nucleophilicity to allow conjugation reactions to take place. It is therefore possible to label a 2'-amino group with a fluorophore.

Unit Structure:

Unit Molecular Weight:
304.20

References:
Smalley, M. K. and Silverman, S. K. Site-specific fluorescent labeling of large RNA with pyrene, in Current Protocols in Nucleic Acids Chemistry Chapter 11, Unit 11.11 (2004).

2'-Amino-uridine

Modification Code:
2'-N-U

Description:
The thermal stability of duplexes containing 2'-amino-RNA has been determined and it was reported that 2'-amino-C substitutions destabilized by about 4° relative to RNA C. It was also further reported that 2'-amino-RNA linkages are nuclease-resistant. The pKa of the 2'-amino group is quite low at 6.2 but this retains sufficient nucleophilicity to allow conjugation reactions to take place. It is therefore possible to label a 2'-amino group with a fluorophore.

Unit Structure:

Unit Molecular Weight:
305.18

References:
Smalley, M. K. and Silverman, S. K. Site-specific fluorescent labeling of large RNA with pyrene, in Current Protocols in Nucleic Acids Chemistry Chapter 11, Unit 11.11 (2004).

2'-Deoxy-uridine

Modification Code:
dU

Description:
DNA modification has long been the industry standard for modifying siRNA 3'-overhang, while conferring nuclease resistance and preserving siRNA potency. DNA is well-tolerated in the passage strand with little effects on siRNA potency. Only partially DNA substituted guide strands are functional, whereas alternating the DNA modification with 2'-f has created fully substituted, active guide strands.

Unit Structure:

Unit Molecular Weight:
290.17

References:
Elbashir, S. M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 411, 494-498 (2001).
Chiu, Y. L. and Rana, T. M. siRNA function in RNAi: a chemical modification analysis. RNA, 9, 1034-1048 (2003).

2'-OMe-inosine

Modification Code:
mI

Description:
The naturally occurring 2'-O-methyl (2'-OMe) modification is the first and most extensively tested 2'-substitutions; 2'-OMe slightly enhances the binding affinity toward RNA (Tm increase of 0.5-0.7 °C per modification). 2'-OMe modification has been extensively used to generate fully functional siRNAs with higher nuclease resistance, reduced siRNA immunogenicity, and less off-targeting. Inosine serves as a more-or-less universal base, as it is capable of pairing with all four natural nucleotides, though not with equal affinity (I-C >I-A>I-T~I-G>I-I).

Unit Structure:

Unit Molecular Weight:
344.22

References:
Soutschek, J. et al. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature, 432, 173-178 (2004).
Allerson, C. R. et al. Fully 2'-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified small interfering RNA. J. Med. Chem, 48, 901-904 (2005).

5-Aminohexylacrylamino-uridine

Modification Code:
5-LC-N-U

Description:
Amine-modified oligonucleotides react with active succinimidyl ester, isothiocyanate, or sulfonyl chloride derivatives of various ligands, such as fluorescent dyes. 5-Aminohexylacrylamino-U is designed in the way that its long linker arm projects into the major groove of double-stranded DNA/RNA. After conjugation, large molecules (i.e. fluorescent dyes) placed cannot interact with the double stranded oligonucleotide thus no disruption of hybridization.

Unit Structure:

Unit Molecular Weight:
474.41

References:
Ruth, J. L. Oligodeoxynucleotides with reporter groups attached to the base., in Olignucleotides and analogues: a practical approach (Eckstein, F. ed.), Oxford University Press, Oxford, pp. 255-282 (1991).

C18 spacer

Modification Code:
18S

Description:
The spacer C3, C9, and 18 are used to insert a spacer arm in an oligonucleotide. Spacers are used for investigating duplex formation, creating distance between an oligonucleotide and a conjugated Modification. 3'-Spacer may also act as a blocker of exonuclease and polymerase activity at the 3'-terminus. The spacers may be added in multiple additions when a longer spacer is required. Deoxy-abasic spacer or ribo-abasic spacer is used to introduce a stable abasic site within an oligonucleotide.

Unit Structure:

Unit Molecular Weight:
344.30

References:
Durand, M. et al. Circular dichroism studies of an oligodeoxyribonucleotide containing a hairpin loop made of a hexaethylene glycol chain: conformation and stability. Nucleic Acid Res. 18, 6353-6359 (1990).

C3 spacer

Modification Code:
C3

Description:
The spacer C3, C9, and 18 are used to insert a spacer arm in an oligonucleotide. Spacers are used for investigating duplex formation, creating distance between an oligonucleotide and a conjugated Modification. 3'-Spacer may also act as a blocker of exonuclease and polymerase activity at the 3'-terminus. The spacers may be added in multiple additions when a longer spacer is required. Deoxy-abasic spacer or ribo-abasic spacer is used to introduce a stable abasic site within an oligonucleotide.

Unit Structure:

Unit Molecular Weight:
138.06

References:
Wang Y. et al. C3-Spacer-containing circular oligonucleotides as inhibitors of human topoisomerase I. Bioorg. Med. Chem. Lett. 18, 3597-3602 (2008).

C9 spacer

Modification Code:
9S

Description:
The spacer C3, C9, and 18 are used to insert a spacer arm in an oligonucleotide. Spacers are used for investigating duplex formation, creating distance between an oligonucleotide and a conjugated Modification. 3'-Spacer may also act as a blocker of exonuclease and polymerase activity at the 3'-terminus. The spacers may be added in multiple additions when a longer spacer is required. Deoxy-abasic spacer or ribo-abasic spacer is used to introduce a stable abasic site within an oligonucleotide.

Unit Structure:

Unit Molecular Weight:
212.14

References:
Durand, M. et al. Circular dichroism studies f an oligodeoxyribonucleotide containing a hairpin loop made of a hexaethylene glycol chain: conformation and stability. Nucleic Acid Res. 18, 6353-6359 (1990).

rSpacer

Modification Code:
rab

Description:
The spacer C3, C9, and 18 are used to insert a spacer arm in an oligonucleotide. Spacers are used for investigating duplex formation, creating distance between an oligonucleotide and a conjugated Modification. 3'-Spacer may also act as a blocker of exonuclease and polymerase activity at the 3'-terminus. The spacers may be added in multiple additions when a longer spacer is required. Deoxy-abasic spacer or ribo-abasic spacer is used to introduce a stable abasic site within an oligonucleotide.

Unit Structure:

Unit Molecular Weight:
196.09

References:

Vesnaver, G. et al. Influence of abasic and anucleosidic sites on the stability, conformation, and melting behavior of a DNA duplex: correlations of thermodynamic and structure data. Proc. Natl. Acad. Sci. USA 86, 3614-3618 (1989).

Taniho, K. et al. Synthesis and biological properties of chemically modified siRNAs bearing 1-deoxy-D-ribofuranose in their 3'-overhang region. Bioorg. Med. Chem. Lett. 22, 2518-2521 (2012).