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.

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-crosslinking 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.

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

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.

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.

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 ($)
8-Oxo-dA-CE Phosphoramidite	10-1008-90	100 µmole	135.00
	10-1008-02	0.25g	355.00
8-Oxo-dG-CE Phosphoramidite	10-1028-95	50 µmole	177.50
	10-1028-90	100 µmole	355.00
	10-1028-02	0.25g	975.00
5-OH-dC-CE Phosphoramidite	10-1063-90	100 µmole	275.00
	10-1063-02	0.25g	775.00
5-OH-dU-CE Phosphoramidite	10-1053-90	100 µmole	225.00
	10-1053-02	0.25g	675.00
5-Hydroxymethyl-dU-CE Phosphoramidite	10-1093-90	100 µmole	225.00
	10-1093-02	0.25g	675.00
5,6-Dihydro-dT-CE Phosphoramidite	10-1530-90	100 µmole	195.00
	10-1530-02	0.25g	595.00
1-Me-dA-CE Phosphoramidite	10-1501-95	50 µmole	125.00
	10-1501-90	100 µmole	250.00
	10-1501-02	0.25g	750.00
N3-Cyanoethyl-dT	10-1531-90	100 µmole	200.00
	10-1531-02	0.25g	600.00
5,6-Dihydro-dU-CE Phosphoramidite	10-1550-90	100 µmole	195.00
	10-1550-02	0.25g	595.00
Pyrene-dU-CE Phosphoramidite	10-1590-95	50 µmole	105.00
	10-1590-90	100 µmole	210.00
	10-1590-02	0.25g	550.00
Perylene-dU-CE Phosphoramidite	10-1591-95	50 µmole	150.00
	10-1551-90	100 µmole	300.00
	10-1591-02	0.25g	720.00

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 naturally to generate abasic sites. Most commonly, dA sites are hydrolyzed causing depurination and leading to abasic residues. 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 Abasic Phosphoramidite allows oligonucleotide synthesis under standard conditions. Following standard deprotection, the silyl protecting groups of the residue are removed with aqueous acid. (This can be done in conjunction with trityl removal in the last step of a DMT-on purification.) The diol so formed is then treated with aqueous sodium periodate to form an aldehyde plus formaldehyde. The aldehyde then immediately cyclizes to its preferred structure, the abasic cyclic sugar (dR). dSpacer has also been used successfully as a mimic of the highly base-labile abasic site.

Item	Catalog No.	Pack	Price ($)
8-Amino-dG-CE Phosphoramidite	10-1079-95	50 µmole	177.50
(dR Precursor)	10-1079-90	100 µmole	355.00
	10-1079-02	0.25g	975.00
Thymidine Glycol-CE Phosphoramidite	10-1096-95	50 µmole	180.00
	10-1096-90	100 µmole	360.00
	10-1096-02	0.25g	975.00
8-OMe-dG-CE Phosphoramidite	10-1075-95	50 µmole	177.50
	10-1075-90	100 µmole	355.00
	10-1075-02	0.25g	975.00
Abasic Phosphoramidite	10-1924-95	50 µmole	105.00
(dR Precursor)	10-1924-90	100 µmole	210.00
	10-1924-02	0.25g	475.00

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 (nucleotide excision repair) or the dimerization is reversed by photolase enzymes. These lesions have been connected to the formation of squamous cell carcinomas. In addition, humans who lack ability to repair CPD lesions with high efficiency may be genetically predisposed to Xeroderma Pigmentosa (XP), a disease characterized by extreme sensitivity to sunlight and high frequency of skin cancer. Polymerases encountering unrepaired CPD lesions are quite error-prone, leading to incorrect base insertions and subsequent mutations.

Item	Catalog No.	Pack	Price ($)
Cis-syn Thymine Dimer Phosphoramidite	11-1330-95	50 µmole	2100.00
(ABI septum vial is standard vial.	11-1330-90	100 µmole	4200.00
Add E to catalog no. for Expedite vial	11-1330-02	0.25g	10200.00
or V to catalog no. for Expedite V vial)

One of the most studied repair mechanisms is probably the base excision DNA repair pathway. 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 design and synthesis of inhibitors that form stable complexes with DNA glycosylases are essential. Complexes can then be studied biochemically and structurally. Toward this end, 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 ($)
Pyrrolidine-CE Phosphoramidite	10-1915-95	50 µmole	190.00
(PYR)	10-1915-90	100 µmole	380.00
	10-1915-02	0.25g	1085.00

BIOLOGICAL MIMICS

Our products for nonenzymatic ligation, 5’-I-dT-CE Phosphoramidite, and 2,4-Difluorotoluene (F), as a non-polar mimic of thymidine, have been discontinued.

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 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 ($)
3'-dA-CE Phosphoramidite	10-1004-95	50 µmole	177.50
	10-1004-90	100 µmole	355.00
	10-1004-02	0.25g	975.00
3'-dC-CE Phosphoramidite	10-1064-95	50 µmole	177.50
	10-1064-90	100 µmole	355.00
	10-1064-02	0.25g	975.00
3'-dG-CE Phosphoramidite	10-1074-95	50 µmole	177.50
	10-1074-90	100 µmole	355.00
	10-1074-02	0.25g	975.00
3'-dT-CE Phosphoramidite	10-1084-95	50 µmole	177.50
	10-1084-90	100 µmole	355.00
	10-1084-02	0.25g	975.00
3'-dA-CPG	20-2004-01	0.1g	300.00
1 µmole columns	20-2104-41	Pack of 4	600.00
0.2 µmole columns	20-2104-42	Pack of 4	200.00
3'-dC-CPG	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
3'-dG-CPG	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
3'-dT-CPG	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

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.

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.

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. 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.

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.