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 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.

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.

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
5,6-Dihydro-dT-CE Phosphoramidite	10-1530-90	100 µmole	195.00
	10-1530-02	0.25g	595.00
5,6-Dihydro-dU-CE Phosphoramidite	10-1550-90	100 µmole	195.00
	10-1550-02	0.25g	595.00
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

BIOLOGICAL MIMICS

Our product, 5’-I-dT-CE Phosphoramidite, has been moved to the click chemistry section of this WEB site and and 2,4-Difluorotoluene (F), as a non-polar mimic of thymidine, has been discontinued.

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.

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 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/labeling, either in solution or on a support. The short DNA labeling strand may feature one of a variety of different labels, such as a biotin group or a fluorophore.

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. To avoid chain branching during synthesis when using DCI as activator, N6-Me-dA is offered with acetyl protection.

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 ™ 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.

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.

The base analogue tC_nitro 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. tC_nitro is virtually non-fluorescent under normal conditions.

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. 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.

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.