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STRUCTURAL STUDIES

STRUCTURE/ACTIVITY RELATIONSHIP

The following products are used to investigate the effect on the activity of an oligonucleotide when key structural elements are changed. The 7-deaza purine monomers lack groups critical for hydrogen bonding. 7-Deaza-8-aza-A and 7-deaza-8-aza-G (PPG) monomers are isomers of A and G and have similar electron density. Their presence in oligos is slightly stabilizing relative to A and G. Unlike G, PPG does not lead to aggregation and G-rich oligos can be easily prepared and isolated. 5'-Fluorescein oligos with PPG at the 5'-terminus are much less quenched than the equivalent G oligos. As a purine analogue of Thymidine, 7-deaza-2'-deoxyXanthosine (7-deaza-dX) promises to have interesting effects on DNA structure of triplexes. 7-Deaza-dX also forms a non-standard base pair with a 2,4-diaminopyrimidine nucleoside analogue. Standard nucleobases have an unshared pair of electrons that project into the minor groove of duplex DNA. Enzymes that interact with DNA, polymerases, reverse transcriptases, restriction enzymes, etc., may use a hydrogen bond donating group to contact the hydrogen bond acceptor in the minor groove. 3-Deaza-2'-deoxyadenosine is very interesting in that it maintains the ability for regular Watson-Crick hydrogen bonding to T but is lacking the electron pair at the 3-position normally provided by N3.

Item Catalog No. Pack Price ($)
10-1001-95 50µm 177.50
10-1001-90 100µm 355.00
10-1001-02 0.25g 975.00
10-1083-95 50µm 177.50
10-1083-90 100µm 355.00
10-1083-02 0.25g 975.00
10-1021-95 50µm 177.50
10-1021-90 100µm 355.00
10-1021-02 0.25g 975.00
10-1073-95 50µm 197.50
10-1073-90 100µm 395.00
10-1073-02 0.25g 1095.00
10-1076-95 50µm 177.50
10-1076-90 100µm 355.00
10-1076-02 0.25g 975.00
10-1088-95 50µm 177.50
10-1088-90 100µm 355.00
10-1088-02 0.25g 975.00

7-Deaza-dA

7-deaza-8-aza-dA

7-deaza-dG

7-Deaza-8-aza-dG (PPG)

7-deaza-dX

3-deaza-dA

STABILITY NOTES

7-Deaza-dG is unstable to iodine oxidation. Add a maximum of 2 times when using iodine oxidation or use 0.5M (10-camphorsulfonyl)- oxaziridine (CSO) in anhydrous acetonitrile and 3 min. oxidation time. (See Glen Report-Vol.9, No.1, 1996,page 8.)

INTELLECTUAL PROPERTY

The use of PPG is subject to proprietary rights of Epoch Biosciences, Inc. and it is sold under license from Epoch Biosciences, Inc. (1) I.V. Kutyavin, et al., Nucleic Acids Res., 2002, 30, 4952-4959.

STRUCTURE/ACTIVITY RELATIONSHIP (CONT.)

The C-nucleoside 2'-deoxypseudouridine, in contrast to dU, forms stable C:pseudoU-A triplets. 2-Aminopurine lacks groups critical for hydrogen bonding and is a mildly fluorescent base.

Demand for sulfur modified bases continues to expand for investigations of oligonucleotide structure, but primarily for cross-linking purposes. 6-Thio-dG, 4-Thio-dT and 4-thio-dU are very useful modifications for photo cross-linking and photoaffinity labelling experiments. Oligos containing 2-thio-dT are useful in examining protein-DNA interaction by acting as photosensitizing probes. The thiocarbonyl group in 2-thio-dT is especially interesting in that it is available to react with compounds associating with the minor groove of DNA. 2-Amino-A forms a very stable base pair with T containing three hydrogen bonds but the stability of the base pair with 2-thio-T is greatly diminished. Due to steric interactions between the 2-thio group of thymidine and the 2-amino group of 2-amino-A, the base pair contains only a single hydrogen bond. Oligos containing 2-amino-dA and 2-thio-dT exhibit high affinity for natural oligonucleotides but show little affinity for other similar oligos even of a complementary sequence.

Item Catalog No. Pack Price ($)
10-1055-95 50µm 177.50
10-1055-90 100µm 355.00
10-1055-02 0.25g 975.00
10-1046-90 100µm 135.00
10-1046-02 0.25g 355.00
10-1072-95 50µm 177.50
10-1072-90 100µm 355.00
10-1072-02 0.25g 975.00
10-1034-95 50µm 165.00
10-1034-90 100µm 295.00
10-1034-02 0.25g 675.00
10-1052-95 50µm 165.00
10-1052-90 100µm 295.00
10-1052-02 0.25g 675.00
10-1036-95 50µm 165.00
10-1036-90 100µm 295.00
10-1036-02 0.25g 675.00

2'-deoxypseudoU

2-Aminopurine

6-thio-dG

4-Thio-dT

O6-Phenyl-dI

2-Thio-dT

STABILITY NOTES

6-Thio-dG, 4-Thio-dT and 4-thio-dU are protected as the S-cyanoethyl ether which is stable during synthesis and readily removed by ammonium hydroxide. It is critical to add 50mM sodium hydrosulfide (NaSH) to the ammonium hydroxide used for deprotection. Especially if room temperature deprotection is carried out, this technique radically reduces the level of ammonolysis which would lead to undesired aminated bases. Moreover, it is also desirable to remove the cyanoethyl protecting group (1M DBU in acetonitrile, 2-5 h/RT) prior to the ammonium hydroxide cleavage and deprotection.

STRUCTURE/ACTIVITY RELATIONSHIP (CONT.)

8-Amino-dA and 8-amino-dG are useful in triplex formation due to the presence of the additional amino groups.

2'-DeoxyXanthosine (dX) is a naturally occurring nucleoside that may be derived from oxidative deamination of 2'-deoxyGuanosine (dG). dX has a similar bonding pattern to thymidine and it may base pair with dA, with such purine-purine interactions causing duplex distortion. dX also featured in attempts to extend the genetic alphabet with a new base pair of dX and pyrimidine-2,4-diamine nucleoside. dX has also interested researchers in the field of DNA damage and repair since it is a product of nitric oxide-induced mutagenesis.

Item Catalog No. Pack Price ($)
10-1086-95 50µm 177.50
10-1086-90 100µm 355.00
10-1086-02 0.25g 975.00
10-1079-95 50µm 177.50
10-1079-90 100µm 355.00
10-1079-02 0.25g 975.00
10-1537-95 50µm 105.00
10-1537-90 100µm 200.00
10-1537-02 0.25g 420.00

8-Amino-dA

8-Amino-dG

dX

STABILITY NOTES

Synthetic oligonucleotides containing 8-amino-dG must be cleaved and deprotected withammonium hydroxide containing 0.25M 2-mercaptoethanol to avoidoxidative degradation of 8-amino-dG sites.

HALOGENATED NUCLEOSIDES

Brominated and iodinated nucleosides are used in crystallography studies of oligonucleotide structure. They are also photolabile and are used for cross-linking studies to probe the structure of protein-DNA complexes. Antibodies exist to Br-dU and oligonucleotides containing Br-dU can be used as probes.

Item Catalog No. Pack Price ($)
10-1007-90 100µm 115.00
10-1007-02 0.25g 295.00
10-1027-90 100µm 105.00
10-1027-02 0.25g 255.00
10-1080-90 100µm 60.00
10-1080-02 0.25g 160.00
10-1081-90 100µm 135.00
10-1081-02 0.25g 355.00
10-1090-90 100µm 60.00
10-1090-02 0.25g 160.00
10-1091-90 100µm 60.00
10-1091-02 0.25g 160.00
10-1092-90 100µm 135.00
10-1092-02 0.25g 355.00
20-2090-01 0.1g 50.00
  1 µmole columns 20-2090-41 Pack of 4 200.00
  0.2 µmole columns 20-2090-42 Pack of 4 120.00

8-Br-dA

8-Br-dG

5-Br-dC

5-I-dC

5-Br-dU

5-I-dU

5-F-dU

STABILITY NOTES

Oligonucleotides containing abromo or iodo group are prepared conventionally with the exceptionthat deprotection is carried out in ammonium hydroxide at room temperature for 24 hours. Under these conditions, degradation of the halogen group was less than 2%.

Deuterated Nucleosides

Perdeuteration and selective deuteration have been useful approaches for simplification of NMR spectra and for other structural studies of large biomolecules. Driven by the progress in multinuclear multidimensional NMR spectroscopy, deuteration of nucleic acids has especially found wide applications in the NMR studies of these complex molecules in solution. 8-Deutero-2'-deoxyGuanosine phosphoramidite will be of interest to our customers involved in NMR spectroscopy.

Item Catalog No. Pack Price ($)
10-1520-90 100µm 90.00
10-1520-02 0.25g 240.00

8-D-dG

STABILITY NOTES

Synthetic oligonucleotides containing 8-D-dG must be cleaved and deprotected with 25% deuterated ammonium hydroxide for 40 hours at room temperature to minimize deuterium exchange.

DNA Damage/Repair

Cellular DNA is constantly being damaged by oxidation and alkylation, by free radicals, and by ultraviolet and ionizing radiation. The body has therefore evolved a number of repair enzyme systems to excise and repair these lesions. The 8-oxo purine monomers allow investigation of the structure and activity of oligonucleotides containing an 8-oxo mutation which is formed naturally when DNA is subjected to oxidative conditions or ionizing radiation. 5,6-Dihydro pyrimidines are naturally occurring compounds that are structural components of alanine transfer RNA. Dihydrouracil and the hydroxy pyrimidines are major base damage products formed by exposure of DNA to ionizing radiation.

Item Catalog No. Pack Price ($)
10-1008-90 100µm 135.00
10-1008-02 0.25g 355.00
10-1028-95 50µm 177.50
10-1028-90 100µm 355.00
10-1028-02 0.25g 975.00
10-1530-90 100µm 195.00
10-1530-02 0.25g 595.00
10-1550-90 100µm 195.00
10-1550-02 0.25g 595.00
10-1063-90 100µm 275.00
10-1063-02 0.25g 775.00
10-1053-90 100µm 225.00
10-1053-02 0.25g 675.00
10-1093-90 100µm 225.00
10-1093-02 0.25g 675.00

8-oxo-dA

8-oxo-dG

5,6-Dihydro-dT

5,6-Dihydro-dU

5-OH-dC

5-OH-dU

5-Hydroxymethyl-dU

STABILITY NOTES

Synthetic oligonucleotides containing 8-oxo-dG must be cleaved and deprotected with ammonium hydroxide containing 0.25M 2-mercaptoethanol to avoid oxidative degradation of 8-oxo-dG sites.

Oligonucleotides synthesized using 5,6-dihydro-dU or 5,6-dihydro-dT and UltraMILD monomers can be cleaved using either concentrated ammonium hydroxide or 50 mM potassium carbonate in anhydrous methanol. Complete cleavage and deprotection can be accomplished at room temperature in 2-4 hours without damaging either the dihydro-dU or dihydro-dT bases.

See Also

DNA Damage/Repair (CONT.)

8-Amino-G is formed along with 8-oxo-G as the major mutagenic lesions formed in DNA damage caused by 2-nitropropane. 2-Nitropropane is an industrial solvent and a component of paints, dyes and varnishes, and is also present in cigarette smoke. Thymine glycol (5,6-dihydroxy-5,6-dihydrothymine) is formed when thymine is subjected to oxidative stress, including ionizing radiation. Oxidation of the 5,6 double bond of Thymidine generates two chiral centers at C5 and C6. The cis-5R,6S form is generated as the predominant product along with the other diastereomer, the cis-5S,6R form. The presence of thymidine glycol in DNA has significant biological consequences and many organisms possess specific repair enzymes for the excision of this lesion. 2-Aminoimidazolone (Iz) and its hydrolysis product imidazolone (Z) are major oxidation products of G. Access to these two potential lesions is not possible during oligonucleotide synthesis because they are so base-labile. A suitable precursor, 8-methoxy-dG (8-OMe-dG), to dIz has now been described. The conversion of 8-OMe-dG to dIz takes place by irradiation of the oligonucleotide (1 mM) in 50 mM sodium cacodylate buffer, pH 7, in the presence of riboflavin (50 µM) for 2 minutes on a transilluminator (366 nm), under aerobic conditions at 4°C. Surprisingly for a photochemical reaction, the conversion is virtually quantitative.

Hydrolysis of nucleoside residues in DNA occurs to generate abasic sites. Most commonly, dA sites are hydrolyzed causing depurination and leading to abasic residues. For researchers trying to determine if their source of depurination in chemical synthesis of DNA is reagent, fluidics or protocol-based, we offer a depurination-resistant dA monomer. A new chemical method allows the generation of abasic sites in double and single stranded oligonucleotides using very mild specific conditions and with very low probability of side reactions. The original Abasic Phosphoramidite (10-1924) has been discontinued since it exhibits low coupling efficiency and the post-synthesis chemistry is fairly challenging. Abasic II Phosphoramidite1 is the replacement for the preparation of a true abasic site. This product has the advantage of simplicity in that the silyl group is removed post-synthesis using aqueous acetic acid. dSpacer has also been used successfully as a mimic of the highly base-labile abasic site.

Item Catalog No. Pack Price ($)
10-1079-95 50µm 177.50
10-1079-90 100µm 355.00
10-1079-02 0.25g 975.00
10-1096-95 50µm 180.00
10-1096-90 100µm 360.00
10-1096-02 0.25g 975.00
10-1075 1.0g Discontinued
10-1927-95 50µm 80.00
10-1927-90 100µm 150.00
10-1927-02 0.25g 475.00
10-1500-90 100µm Discontinued
10-1500-02 0.25g Discontinued

8-Amino-dG

Thymidine Glycol CE

8-OMe-dG

Abasic II

dbf-dA

STABILITY NOTES

Synthetic oligonucleotides containing 8-oxo-dG must be cleaved and deprotected with ammonium hydroxide containing 0.25M 2-mercaptoethanol to avoid oxidative degradation of 8-oxo-dG sites.

Oligonucleotides synthesized using 5,6-dihydro-dU or 5,6-dihydro-dT and UltraMILD monomers can be cleaved using either concentrated ammonium hydroxide or 50 mM potassium carbonate in anhydrous methanol. Complete cleavage and deprotection can be accomplished at room temperature in 2-4 hours without damaging either the dihydro-dU or dihydro-dT bases.

  1. K. Groebke, and C.J. Leumann, Helv Chim Acta, 1990, 73, 608-617.

See Also

8-oxo Iz                  Z

DNA Damage/Repair (CONT.)

One of the major sources of DNA damage in all organisms is the UV component of sunlight. The predominant reaction induced by UV light on DNA is dimerization of adjacent pyrimidine bases leading to cyclobutane dimers (CPDs). The dimers formed in the most significant quantity are the cis-syn cyclobutane dimer of two thymine bases. Although formed routinely, these dimer products are efficiently excised and repaired enzymatically by nucleotide excision repair (NER) or the dimerization is reversed by photolase enzymes. A further mode of oxidative damage is radiation-induced damage of DNA, which has been shown to lead to bridged cyclonucleosides. The purines, cyclo-dA and cyclo-dG, are predominantly formed, although the cyclo pyrimidines have also been detected. Cyclo-dA is doubly intriguing since it contains both damaged base and damaged sugar residues and, as such, should have a considerable biological impact. In a manner analogous to thymine dimer, cyclo purines cause significant distortion of the regular DNA helix and these lesions are repaired not by base excision repair (BER) but by NER.

Item Catalog No. Pack Price ($)
11-1330-95 50µm 2100.00
11-1330-90 100µm 4200.00
11-1330-02 0.25g 10200.00
10-1098-95 50µm 950.00
10-1098-90 100µm 1850.00
10-1098-02 0.25g 5350.00
10-1598-95 50µm 1250.00
10-1598-90 100µm 2450.00

Cis-syn Thymine Dimer

8,5'-Cyclo-dA CE

8,5'-Cyclo-dG

INSTRUMENT TYPES

For these very expensive phosphoramidites, an ABI septum vial is the standard vial. Add E to the catalog no. for an Expedite vial or V to the catalog no. for an Expedite V vial.

DNA Damage/Repair (CONT.)

Base excision repair (BER) is one of the most studied repair mechanisms. In this pathway, DNA glycosylases recognize the damaged bases and catalyze their excision through hydrolysis of the N-glycosidic bond. Attempts to understand the structural basis for DNA damage recognition by DNA glycosylases have been hampered by the short-lived association of these enzymes with their DNA substrates. To overcome this problem, the Verdine group at Harvard synthesized a pyrrolidine analog that mimics the charged transition state of the enzyme-substrate complex. When incorporated into double-stranded DNA, they found the pyrrolidine analog (PYR), introduced as the Pyrrolidine-CE Phosphoramidite, forms an extremely stable complex with the DNA glycosylase AlkA, exhibiting a dissociation constant in the pM range and potently inhibited the reaction catalyzed by the enzyme.

Item Catalog No. Pack Price ($)
10-1915-95 50µm 190.00
  (PYR) 10-1915-90 100µm 380.00
10-1915-02 0.25g 1085.00

Pyrrolidine

CLICK DNA AND RNA LIGATION

Ligation of an oligo containing a 5'-azide with an oligo containing a 3'-propargyl group using Click Chemistry leads to a triazole linkage that has been shown to have in vivo biocompatibility. This technique has been used to synthesize DNA constructs up to 300 bases in length. When the resultant triazole linkage was placed in a PCR template, various polymerases were able to copy the sequence correctly. The linkage has also been shown to be compatible with transcription and rolling circle amplification, as well as gene expression in E. coli. In the RNA world, a hammerhead ribozyme containing the triazole linkage at the substrate cleavage site has been shown to retain its activity. A large variety of applications is envisaged for this biocompatible chemical ligation. Support for this technology is offered with the help of Tom Brown's group at the University of Southampton.

Item Catalog No. Pack Price ($)
20-2982-01 0.1g 180.00
20-2982-10 1.0g 1500.00
  1 µmole columns 20-2982-41 Pack of 4 300.00
  0.2 µmole columns 20-2982-42 Pack of 4 150.00
  10 µmole column (ABI) 20-2982-13 Pack of 1 750.00
  15 µmole column (Expedite) 20-2982-14 Pack of 1 1125.00

3'-Propargyl-5-Me-dC CPG

Biocompatible Triazole Linkage

BROWN

See Also

REFERENCES - CLICK LIGATION

  1. A.H. El-Sagheer, A.P. Sanzone, R. Gao, A. Tavassoli, and T. Brown, Proc Natl Acad Sci U S A, 2011, 108, 11338-43.
  2. A.H. el-Sagheer, and T. Brown, Chem Commun (Camb), 2011, 47, 12057-8.
  3. A.P. Sanzone, A.H. El-Sagheer, T. Brown, and A. Tavassoli, Nucleic Acids Res, 2012.
  4. A. Dallmann, et al., Chemistry, 2011, 17, 14714-7.
  5. A.H. El-Sagheer, and T. Brown, Proc Natl Acad Sci U S A, 2010, 107, 15329-34.

5'-LABELlING OF MicroRNAs

Several methods have been developed for the detection of miRNAs, however, few allow the simultaneous detection of multiple miRNAs. To overcome this analytical deficiency, the Richert group at the University of Stuttgart has recently developed an ingenious method to selectively detect miRNAs on microarrays without interference from endogenous pre-mRNAs, mRNAs and other RNA species. In this method, a short oligonucleotide containing 3'-amino-dT and a 5' reporter molecule is chemically ligated to the microRNA in a one-step procedure by in situ activation of the microRNA. This is specifically achieved by taking advantage of the fact that miRNAs, unlike other RNAs, are 5'-phosphorylated. The reaction is template-directed (and thus sequence specific) and can be performed together with enzymatic 3'-extension/labelling, either in solution or on a support. The short DNA labelling strand may feature one of a variety of different labels, such as a biotin group or a fluorophore.

Item Catalog No. Pack Price ($)
20-2981-01 0.1g 120.00
20-2981-10 1.0g 995.00
  1 µmole columns 20-2981-41 Pack of 4 200.00
  0.2 µmole columns 20-2981-42 Pack of 4 120.00
  10 µmole column (ABI) 20-2981-13 Pack of 1 500.00
  15 µmole column (Expedite) 20-2981-14 Pack of 1 750.00

3'-Amino-dT CPG

REFERENCES - MicroRNA LABELLING

  1. H. Vogel, and C. Richert, ChemBioChem, 2012, 13, 1474-82.
  2. R. Eisenhuth, and C. Richert, Journal of Organic Chemistry, 2008, 74, 26- 37.
  3. E . Kervio, A. Hochgesand, U.E. Steiner, and C. Richert, Proc Natl Acad Sci U S A, 2010, 107, 12074-9.

2'-5' Linked Oligonucleotides

Cellular DNA and RNA are made up of ribo- and 2'-deoxyribonucleic acids linked together via 3'-5' phosphodiester linkages and by far comprise the bulk of polynucleic acids found in cells. Much less common are oligonucleotides which have 2'-5' linkages. However, a unique feature of 2'-5' linked oligonucleotides is their ability to bind selectively to complementary RNA. These features suggest a number of interesting uses for 2'-5' linked oligos such as their use as RNA specific probes or in antisense oligos. Recently, oligos have been synthesized using 3'-deoxy-2'-phosphoramidites and 2'-deoxy-3'-phosphoramidites to produce chimeras with 2'-5' linked ends and 3'-5' linked central regions. It was found that 2'-5' phosphorothioate oligos: 1) bind selectively to complementary RNA with the same affinity as phosphodiester oligos; 2) exhibit much less nonspecific binding to cellular proteins; 3) do not activate RNase H. A 3'-deoxynucleoside at the 3'-terminus of an otherwise normal oligonucleotide effectively blocks polymerase extension.

Item Catalog No. Pack Price ($)
10-1004-95 50µm 177.50
10-1004-90 100µm 355.00
10-1004-02 0.25g 975.00
10-1064-95 50µm 177.50
10-1064-90 100µm 355.00
10-1064-02 0.25g 975.00
10-1074-95 50µm 177.50
10-1074-90 100µm 355.00
10-1074-02 0.25g 975.00
10-1084-95 50µm 177.50
10-1084-90 100µm 355.00
10-1084-02 0.25g 975.00
20-2004-01 0.1g 400.00
  1 µmole columns 20-2104-41 Pack of 4 675.00
  0.2 µmole columns 20-2104-42 Pack of 4 225.00
20-2064-01 0.1g 300.00
  1 µmole columns 20-2164-41 Pack of 4 600.00
  0.2 µmole columns 20-2164-42 Pack of 4 200.00
20-2074-01 0.1g 300.00
  1 µmole columns 20-2174-41 Pack of 4 600.00
  0.2 µmole columns 20-2174-42 Pack of 4 200.00
20-2084-01 0.1g 300.00
  1 µmole columns 20-2184-41 Pack of 4 600.00
  0.2 µmole columns 20-2184-42 Pack of 4 200.00

3'-dA

3'-dC

3'-dG

3'-dT

3'-dA-CPG

3'-dC-CPG

3'-dG-CPG

3'-dT-CPG

See Also

MUTAGENESIS

Cellular polynucleotides are alkylated by endogenous components, such as S-adenosylmethionine, or after reacting with two general classes of environmental and laboratory chemicals. SN1 chemical agents include alkylnitrosourea and N-alkyl-N-nitro-N-nitrosoguanidine that react with the N7 position of guanine, N3 of adenine, O6 of guanine, O2 or O4 of pyrimidines, and the non-phosphodiester oxygen atoms of the phosphate backbone. In contrast, SN2 chemical agents such as methyl methanesulfonate and dimethyl sulfate react primarily with the N1 position of adenine (1-Methyl-2'-deoxyadenosine) and N3 of cytosine. To avoid chain branching during synthesis when using DCI as activator, N6-Me-dA is offered with acetyl protection.

Item Catalog No. Pack Price ($)
10-1070-90 100µm 105.00
10-1070-02 0.25g 255.00
10-1003-90 100µm 162.50
10-1003-02 0.25g 495.00
10-1503-90 100µm 162.50
10-1503-02 0.25g 495.00
10-1032-90 100µm 135.00
10-1032-02 0.25g 355.00
10-1501-95 50µm 125.00
10-1501-90 100µm 250.00
10-1501-02 0.25g 750.00

O6-Me-dG

N6-Me-dA

N6-Ac-N6-Me-dA

O4-Me-dT

1-Me-dA

See Also

CONVERTIBLE NUCLEOSIDES

The convertible nucleoside strategy is one of the most versatile methods for producing modifications in bases to examine their effects on DNA structure and activity. In some cases, with versatility comes difficulty in that the convertible base is modified after oligonucleotide synthesis. The chemistry is sometimes complex and base composition analysis of the final oligonucleotide is required to verify structure. The convertible dU monomer can be used to introduce a variety of modifications at the convertible position, including N, O and S modifications. Convertible F-dC is by far the simplest approach to the preparation of oligonucleotides containing F-dC - normal ammonium hydroxide treatment effects the conversion to F-dC. Convertible dA has been used to prepare oligonucleotides containing multiple points for attachment to solid supports. In this way, high capacity affinity supports for the purification of DNA binding proteins have been prepared. 2-F-dI is a convertible nucleoside for the preparation of 2'-dG derivatives following the displacement of the 2-fluorine by primary amines.

Item Catalog No. Pack Price ($)
10-1016-90 100µm 195.00
  (Convertible F-dC) 10-1016-02 0.25g 495.00
10-1042-90 100µm 135.00
  (Convertible dA) 10-1042-02 0.25g 355.00
10-1051-90 100µm 135.00
  (Convertible dU) 10-1051-02 0.25g 355.00
10-1082-95 50µm 180.00
  (Convertible dG) 10-1082-90 100µm 360.00
10-1082-02 0.25g 975.00

TMP-F-dU

O6-Phenyl-dI

O4-Triazolyl-dU

2-F-dI

ABBREVIATION

TMP = 2,4,6-trimethylphenyl

FLUORESCENT NUCLEOSIDES

Etheno-dA is a fluorescent nucleoside which is especially useful in observing the transition between DNA structural types. It is quite base labile and should be deprotected with ammonium hydroxide at room temperature for 24 hours. Alternatively, UltraMild chemistry can be used. 2-Aminopurine and AP-dC (G-Clamp) are also useful fluorescent nucleosides.

Pyrrolo-dC is a fluorescent deoxycytidine analog that is an ideal probe of DNA structure and dynamics.1,2 It base-pairs as a normal dC nucleotide. An oligo fully substituted with pyrrolo-dC has the same Tm as the control dC oligo with the same specificity for dG. Its small size does not perturb the structure of the DNA helix and it is well tolerated by a number of DNA and RNA polymerases. It is highly fluorescent and its excitation and emission are well to the red of most fluorescent nucleotide analogs, which eliminates or reduces background fluorescence from proteins. Pyrrolo-dCTP has potential uses in biological assay development.

Item Catalog No. Pack Price ($)
10-1006-90 100µm 105.00
10-1006-02 0.25g 255.00
10-1017-95 50µm 110.00
10-1017-90 100µm 220.00
10-1017-02 0.25g 675.00
81-1017-01 0.1mL 150.00

Etheno-dA

Pyrrolo-dC

Pyrrolo-dCTP (10mM)

See Also

INTELLECTUAL PROPERTY

Pyrrolo-dC is a joint development project of Berry & Associates, Inc. and Glen Research Corporation. Pyrrolo-dC is covered by US Patent No.: 7,144,995.

SPECTRAL PROPERTIES

The spectral properties of pyrrolodC, coupled with its unique base-pairing ability, make this fluorescent analog extremely valuable in probing DNA structure. When the pyrrolo-dC is base-paired, its fluorescence is significantly quenched through what is most likely base stacking or dG interactions. The quantum yield of fluorescence for pyrrolo-dC is quite sensitive to its hybridization state, making it ideally suited for probing the dynamic structure of DNA.

QY
  
 (L/mol.cm)
single-
stranded
0.07 260nm 4000
  347nm 3700
double-
stranded
0.02   

(QY determined relative to quinine sulfate in 0.5M H2SO4)

References

  1. D.A. Berry, et al., Tetrahedron Lett, 2004, 45, 2457-2461.
  2. The Glen Report, 2007, 19, 8-9.
  3. P. Sandin, et al., Nucleic Acids Res., 2008, 36, 157-167.
  4. P. Sandin, et al., Nucleic Acids Res., 2005, 33, 5019-5025.
  5. K.C. Engman, et al., Nucleic Acids Res., 2004, 32, 5087-5095.

FLUORESCENT NUCLEOSIDES (Cont.)

When a benzylcarbamoyl analogue of AP-dC (G-Clamp) was synthesized, it was found that, when incorporated into an oligo, it exhibited similar fluorescence to AP-dC. However, when base-paired against the 8-oxo-dG, its fluorescence was severely quenched. Rather remarkably, however, when base paired with dG or any of the other bases, A, C or T, there was no change in fluorescence - making it a specific probe for 8-oxo-dG.

By attaching pyrene or perylene to the 5 position of deoxyuridine through a triple bond, the fluorophore is electronically coupled to the deoxyuridine base. This electronic coupling of the base and the fluorophore makes the fluorescence sensitive to the base pairing of the dU portion of the molecule, allowing the discrimination between perfect and one base mismatched targets.

Item Catalog No. Pack Price ($)
10-1515-95 50µm 260.00
10-1515-90 100µm 500.00
10-1515-02 0.25g 1220.00
10-1590-95 50µm 105.00
10-1590-90 100µm 210.00
10-1590-02 0.25g 550.00
10-1591-95 50µm 150.00
10-1591-90 100µm 300.00
10-1591-02 0.25g 720.00

8-oxo-G Clamp CE-Phosphoramidite

Pyrene-dU

Perylene-dU

See Also

SPECTRAL PROPERTIES

Absorbance
Maximum
Emission
Maximum
Pyrene-dU 402nm 472nm
Perylene-dU 473nm 490nm

FLUORESCENT NUCLEOSIDES (CONT.)

The tricyclic fluorescent nucleoside analogues, 1,3-diaza-2-oxophenothiazine, tC, and 1,3-diaza-2-oxophenoxazine, tCo, are deoxycytidine analogs that have been shown to base pair faithfully with dG with virtually no disruption of the normal duplex structure.3-5 This means that the stability of the DNA duplex is not compromised as compared to the control regardless of DNA sequence. The fluorescence quantum yield of tC is essentially unchanged between single stranded and double stranded DNA - 0.21 for single stranded DNA and 0.19 for duplex DNA. Also, the fluorescence characteristics of tC are not sensitive to neighboring base combinations. tCo has been shown to be the brightest fluorescent nucleoside analogue in duplex context reported so far and even retains the majority of its fluorescence when surrounded by guanine residues. Indeed, tCo has been reported to be 25-50 times brighter than 2-aminopurine.

The base analogue tCnitro is a FRET-acceptor together with tCO (or tC) as the donor molecule. This constitutes the first ever description of a nucleobase FRET-pair. This novel FRET-pair provides a unique tool for investigations of nucleic acid containing systems. tCnitro is virtually non-fluorescent under normal conditions.

Item Catalog No. Pack Price ($)
10-1516-95 50µm 250.00
10-1516-90 100µm 490.00
10-1516-02 0.25g 1460.00
10-1517-95 50µm 250.00
10-1517-90 100µm 490.00
10-1517-02 0.25g 1460.00
10-1518-95 50µm 265.00
10-1518-90 100µm 520.00
10-1518-02 0.25g 1460.00

tC

tC°

tCnitro

SPECTRAL PROPERTIES

Absorption and emisssion data for tC and tCo are collected below:

tC QY
      (L/mol.cm)
single-
stranded
0.21 385nm 4000
double-
stranded
0.19    
tCo QY
      (L/mol.cm)
single-
stranded
0.30 360nm 9000
double-
stranded
0.21    

(QY determined relative to quinine sulfate in 0.5M H2SO4)

INTELLECTUAL PROPERTY

These products are offered under license from Isis Pharmaceuticals, Inc. and in collaboration with ModyBase HB.

Caged NUCLEOSIDES

Glen Research's interest lies in the preparation of caged oligonucleotides whose function is restored after uncaging by UV light at a wavelength that causes no DNA damage. The Deiters group at North Carolina State University has described NPOM-Caged-dT, where the nucleobase is caged with the photolabile group, 6-nitropiperonyloxymethyl (NPOM), which can be removed using UV light at 365nm. Oligonucleotides containing NPOM-Caged-dT every five or six bases do not hybridize to their complementary strand. Photo-uncaging of the caged oligonucleotide is then easily carried out with UV light at 365 nm for seconds to minutes to restore the activity of the oligonucleotide.

Item Catalog No. Pack Price ($)
10-1534-95 50µm 185.00
10-1534-90 100µm 355.00
10-1534-02 0.25g 895.00

N-POM Caged-dT

THERAPEUTIC Nucleosides

Cytosine Arabanoside (Ara-C) is an anti-viral drug which has achieved limited use. Its effect on DNA structure and activity can be investigated by incorporating it into synthetic oligonucleotides.

Zebularine (pyrimidin-2-one ribonucleoside) is a cytidine analogue that acts as a DNA demethylase inhibitor, as well as a cytidine deaminase inhibitor. This structure is very active biologically and Zebularine is now used as a potent anti-cancer drug. A 2'-deoxynucleoside analogue of Zebularine, 5-methyl-pyrimidin-2-one, 2'-deoxynucleoside, has been used to probe the initiation of the cellular DNA repair process by making use of its mildly fluorescent properties. This combination of biological activity and fluorescence properties would make 5-Me-2'-deoxyZebularine a strong addition to our array of nucleoside analogues.

Cytosine-5-methyltransferases are found in everything from archaebacteria to mammals and when the regulation of cytosine-5-methyltransferases goes awry, cancer can result. The mechanism of action for this family of enzymes involves attack of a cysteine thiol group on the C6 position of cytosine, leading to a transient dihydrocytosine intermediate, which then facilitates the nucleophilic attack by C5 on the activated methyl group of the S-adenosyl-L-methionine cofactor. As with many enzymes, the intermediate can be trapped using a suicide substrate and 5-fluoro-cytosine has been used extensively in this role. An alternate strategy is to use a transition-state mimic that binds to the active site with high affinity. An excellent candidate was found in 5-aza-5,6-dihydrocytosine. Despite not being covalently bound to the enzyme, it was found1,2 to be a more potent inhibitor of cytosine-5-methyltransferases than 5-fluoro-cytosine. 5-Aza-5,6-dihydro-dC is compatible with standard oligonucleotide synthesis and deprotection conditions and is an excellent tool for use in methyltransferase research.

Item Catalog No. Pack Price ($)
10-4010-90 100µm Discontinued
10-4010-02 0.25g Discontinued
10-1061-95 50µm 200.00
10-1061-90 100µm 400.00
10-1061-02 0.25g 975.00
10-1511-95 50µm 180.00
10-1511-90 100µm 360.00
10-1511-02 0.25g 1120.00

Ara-C

5-Me-2'-deoxyZebularine

5-aza-5,6-dihydro-dC

 

References

  1. G. Sheikhnejad, et al., J Mol Biol, 1999, 285, 2021-2034.
  2. V .E. Marquez, et al., Antisense Nucleic Acid Drug D, 1999, 9, 415-421.

See Also

LARGE SCALE SYNTHESIS

The most common side reaction during deprotection of oligonucleotides on a large scale is the alkylation of dT residues by acrylonitrile, formed by β-elimination of the cyanoethyl phosphate protecting groups, to generate N3-cyanoethyl-dT.

Item Catalog No. Pack Price ($)
10-1531-90 100µm 200.00
10-1531-02 0.25g 600.00

N3-Cyanoethyl-dT

 

 

Please contact Glen Research if you have any questions or comments!
 
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