Serinol Reagents for Modification and Labelling

Most popular non-nucleosidic phosphoramidites for modification and labelling are based on two structural types: 1,2-diols and 1,3-diols. Products based on a 1,2-diol backbone were first described to allow amino-modification and biotin labelling. Technically, the 1,2-diol backbone has some drawbacks relative to the 1,3-diol backbone. The 1,2-diol backbone can participate in a dephosphorylation reaction since the 1,2-diol can form a favored 5-membered cyclic phosphate intermediate. This reaction is competitive with simple hydrolysis of the protecting groups and leads to some loss of label. However, the degree of loss at the 3’ terminus can be limited by the removal of the cyanoethyl protecting group using DBU or diethylamine prior to the cleavage and deprotection steps. Similarly, loss at the 5’ terminus can be eliminated by retaining the DMT group until the oligo is fully deprotected. Fortunately, the elimination reaction is virtually non-existent in the 1,3-diol backbone since the cyclic intermediate would be a 6-membered ring which is not favored for a cyclic phosphate intermediate.

IVD customers have requested a new backbone based on a 1,3-diol that would overcome any technical or IP issues surrounding our current products. We now offer a line of products based on the serinol backbone, which have been developed in close collaboration between Glen Research and Nelson Biotechnologies. Protected Biotin Serinol Phosphoramidite and CPG are protected with a t-butylbenzoyl group on the biotin ring. This group is designed to stop any phosphoramidite reactions at this active position in biotin. This protection avoids branching when using nucleophilic activators like DCI. The protecting group is easily removed during oligonucleotide cleavage and deprotection. The BiotinLC versions are similarly protected and should be useful for the synthesis of highly sensitive biotinylated probes. 6-Fluorescein Serinol Phosphoramidite and CPG are designed to prepare oligonucleotides containing one or several 6-Fluorescein (6-FAM) residues. Amino-Modifier Serinol Phosphoramidite and CPG are used to add amino groups into one or several positions in oligonucleotides. The amino group is protected with Fmoc, which may be removed on the synthesis column prior to solid-phase conjugation to the amino groups, or which may be removed during deprotection for subsequent solution phase conjugation to the amino groups.

BIOTIN LABELLING

Biotin-dT can replace dT residues within the oligonucleotide sequence. 5’-Biotin phosphoramidite can be added ONLY ONCE to the 5’-terminus of an oligonucleotide. However, the DMT group on the biotin can be used in RP cartridge and HPLC purification techniques. PC Biotin is a photocleavable 5’-biotin phosphoramidite. BiotinTEG CPG and Protected BiotinLC Serinol CPG are designed for the direct synthesis of oligonucleotides containing biotin at the 3’ terminus.

Desthiobiotin is a biotin analogue that exhibits lower binding to biotin-binding proteins such as streptavidin. This biotin analogue is lacking the sulfur group from the molecule and has a dissociation constant (Kd) several orders of magnitude less than biotin/streptavidin. As a result, biomolecules containing desthiobiotin are dissociated from streptavidin simply in the presence of buffered solutions of biotin. We offer desthiobiotinTEG phosphoramidite and the corresponding CPG.

ABI-style vials and columns are supplied unless otherwise requested (see note box).

FLUORESCEIN LABELLING

5’-Fluorescein phosphoramidite contains no 4,4’-dimethoxytrityl (DMT) group and can be added only once at the 5’-terminus, thereby terminating synthesis. This product is prepared using the 6-carboxyfluorescein derivative. The tetrachloro-, hexachloro-and dichloro-dimethoxy-fluorescein phosphoramidites are designed to take advantage of the multicolor detection capability of modern DNA sequencers and genetic analyzers. Fluorescein phosphoramidite is designed to produce the same fluorescein-type structure as had been previously prepared using fluorescein isothiocyanate (FITC). Our fluorescein phosphoramidite also contains a DMT group to allow quantification of coupling. The analogous structure, 6-Fluorescein Phosphoramidite, prepared using 6-FAM, is also available, along with 6-Fluorescein Serinol Phosphoramidite. Fluorescein-dT can be inserted into the desired sequence as a replacement for a dT residue.

We offer five fluorescein supports. Fluorescein CPG has traditionally been used to add the fluorescein label at the 3’-terminus. The analogous structure, 3’-(6-Fluorescein) CPG, prepared using 6-FAM, is now also available, along with 6-Fluorescein Serinol CPG. We also offer 3’-(6-FAM) CPG and Fluorescein-dT CPG, both derivatives of 6-carboxyfluorescein (6-FAM). Both are single isomers and use an amide linkage which is stable during cleavage and deprotection and does not allow isomer formation. 3’-(6-FAM) CPG allows effective blockage of the 3’-terminus from polymerase extension as well as exonuclease digestion. Fluorescein-dT CPG allows both of these enzymatic activities to proceed. Normal cleavage and deprotection with ammonium hydroxide readily generates the fluorescein labelled oligos.

The spectral characteristics of these dyes are detailed on the right side of this page.

Fluorescein Laelling (SIMA)

Dichloro-diphenyl-fluorescein, SIMA (HEX) exhibits virtually identical absorbance and emission spectra to HEX. SIMA (HEX) is much more stable to basic deprotection conditions than HEX and oligonucleotides can be deprotected using ammonium hydroxide at elevated temperatures and even ammonium hydroxide/methylamine (AMA) at room temperature or 65°C for 10 minutes. SIMA absorption maximum was 3 nm blue-shifted compared to HEX at pH 7. The absorbance is broader, so the extinction coefficient is smaller than that of HEX, but when exciting at 500 nm where the absorbance was normalized, the emission was still 90% of HEX and the emission was red-shifted by 5 nm. A second SIMA (HEX) product, SIMA (HEX)-dT, can be used to introduce SIMA (HEX) in the synthetic oligonucleotide sequence, usually as a replacement for the native dT linkage. Again, this product is fully compatible with deprotection schemes using ammonium hydroxide at elevated temperatures or AMA at room temperature and 65°C.

RHODAMINE LABELLING

Rhodamine derivatives are not sufficiently stable to survive conventional deprotection and these must be attached to amino-modified oligonucleotides using post-synthesis labelling techniques. Because Tetramethyl Rhodamine (TAMRA) is not base stable, the procedure to cleave and deprotect the labelled oligonucleotide must be carefully considered. Using the UltraMILD monomers and deprotection with potassium carbonate in methanol, TAMRA oligonucleotides can be fairly conveniently isolated. To streamline the preparation of TAMRA oligos, we offer 3’-TAMRA CPG for 3’ labelling and TAMRA-dT for labelling within the sequence. We also offer TAMRA NHS ester for labelling amino-modified oligonucleotides.

CYANINE LABELLING

Two cyanine derivatives, Cy3™ and Cy5™, which differ in structure simply by the number of carbons in the conjugated poly-ene linkage, are joined by the closely related analogues, Cy3.5™ and Cy5.5™, and are available as phosphoramidites. Cy dyes are normally added once at the 5’-terminus and the MMT group should be removed on the synthesizer. The absorbance of the MMT cation (yellow) is noticeably different from the DMT cation (orange), and so, absorbance-based trityl monitors will detect it incorrectly as a low coupling. On the other hand, conductivity detectors will interpret the release more correctly. Cy dyes have also been used successfully adjacent to the 3’-terminus as long as 0.02M iodine oxidizer is used during the remainder of the synthesis.

Deprotection of oligos containing Cy dyes may be carried out with ammonium hydroxide at room temperature, regardless of the base protecting groups on the monomers used. If there is a need to use ammonium hydroxide at elevated temperature, Cy3 and Cy 3.5 are more stable than Cy5 and Cy5.5. However, it is always prudent to use monomers with base labile protecting groups to limit the exposure time to 2 hours or less at 55°C.

*Note price increase as of 1/14/2010

DyLight™ Dyes

DyLight™ dyes, DY547 and DY647, are phosphoramidite alternatives to Cy3 and Cy5. The performance of these two dyes is similar to the equivalent Cy dyes and the absorption and emission spectra are virtually identical. A comparison of the emission or absorbance spectra of oligos shows that the spectra are virtually identical for Cy3 and DY547. Similarly, Cy5 and DY 647 are virtually identical. When oligos containing Dy547 were excited at 488 nm, the quantum yield (QY) was 12% greater than oligos containing Cy3. When oligos containing Dy647 were excited at 580 nm, the QY was 5% greater and the emission was blue-shifted by only 1 nm compared to Cy5.

EPOCH DYES AND QUENCHER

Glen Research’s agreement with Epoch Biosciences, Inc., a subsidiary of Nanogen, Inc., allows us to sell several of Epoch’s proprietary products designed for the synthesis of novel DNA probes. We are pleased to offer products based on Epoch’s Redmond Red™, Yakima Yellow™ and Gig Harbor Green™ fluorophores and Eclipse™ non-fluorescent quencher. Under our agreement we also supply PPG, a modified nucleoside and 5’-Aldehyde-Modifier C2 Phosphoramidite. The two fluorescent dyes, Yakima Yellow and Redmond Red, are available as phosphoramidites and supports. Yakima Yellow has an absorbance maximum at 530 nm and emission maximum at 549 nm, while Redmond Red’s absorbance and emission maxima are at 579 nm and 595 nm, respectively. Gig Harbor Green and 6-FAM are based on the same fluorescein core structure but Gig Harbor Green is 15-20% brighter than FAM.

The Eclipse quencher from Epoch solves most of the problems inherent in the synthesis of molecular beacon and FRET probes. The Eclipse molecule is highly stable and can be used safely in all common oligo deprotection schemes. The absorbance maximum for Eclipse Quencher is at 522 nm, compared to 479 nm for dabcyl. In addition, the structure of the Eclipse Quencher is substantially more electron deficient than that of dabcyl and this leads to better quenching over a wider range of dyes, especially those with emission maxima at longer wavelengths (red shifted) such as Redmond Red and Cy5. In addition, with an absorption range from 390 nm to 625 nm, the Eclipse Quencher is capable of effective performance in a wide range of colored FRET probes.

Black Hole Quencher Dyes

With the growing popularity of red and near-infrared dyes, we are offering the Black Hole QuencherTM dyes (BHQs), whose physical properties are detailed in Table 1. BHQ dyes are robust dark quenchers that very nicely complement our existing product line. They are compatible with ammonium hydroxide deprotection, exhibit excellent coupling efficiencies, have large extinction coefficients and are completely non-fluorescent. Their absorbances are well-tuned to quench a variety of popular fluorophores – even those far into the red, such as Cy3 and Cy5. The dark quencher most typically used in a Molecular Beacon is Dabcyl. Because the quenching does not involve FRET, there is little, if any, dependence upon donor-acceptor spectral overlap. In a comprehensive paper by Marras, Kramer and Tyagi,¹ the ability of BHQ-1 and BHQ-2 to quench 22 different fluorophores was evaluated. For shorter wavelength fluorophores such as fluorescein, the quenching efficiency was roughly the same as Dabcyl (91% – 93%). However, for dyes emitting in the far red, such as Cy5, the BHQ dyes were far superior – quenching the Cy5 with 96% efficiency, compared to 84% with Dabcyl. This may reflect the BHQ’s ability to form stable, non-fluorescent complexes which can be a plus even in FRET probes. Indeed, recent work suggests that these non-fluorescent complexes will form even in the absence of a hairpin stem structure used by Molecular Beacons.²

BlackBerry® Quencher (BBQ-650®)

We are happy to offer several products containing the BlackBerry® Quencher (BBQ-650®), which exhibits a broad absorption profile from 550nm to 750nm, centered at 650nm. This range offers more effective quenching of some of our popular long wavelength dyes like TAMRA, Redmond Red, Cy dyes and DyLight dyes. We offer BBQ-650 products for the 3' and 5' termini, as well as BBQ-650-dT for inclusion within the oligonucleotide sequence, with the following properties:

• Quenches the fluorescence of long wavelength dyes
• Quenches in FRET and contact mode
• Absorbance maximum at ~650nm
• Quenching range - 550-750nm
• Compatible with standard oligo synthesis chemistry>
• Compatible with regular deprotection but requires mild deprotection with AMA at room temperature
• Available for 3’, 5’, and internal substitution

acridine LABELLING

Acridine phosphoramidite is designed to produce an oligonucleotide containing acridine at any position in the molecule. Acridine CPG is used to label the 3’-terminus. Acridine is an effective intercalating agent.

DNP LABELLING

An analytical test based on detection of 2,4-dinitrophenyl (DNP) labelled oligonucleotides with anti-DNP antibodies has been proposed. We have chosen the branched triethylene glycol (TEG) spacer in our version of DNP phosphoramidite since it can be added once or several times to the 3’ or 5’ terminus.

CHOLESTEROL LABELLING

Potential therapeutic oligonucleotides must permeate the cell membrane for optimal activity. The addition of lipophilic groups to an oligonucleotide would be expected to enhance activity. The use of cholesteryl oligos and the consequent improvement in activity has been described. We have designed our Cholesteryl Phosphoramidite with our branched triethyleneglycol (TEG) spacer for maximum solubility in acetonitrile as well as for applications requiring multiple labels.

ABI-style vials and columns are supplied unless otherwise requested (see note box).

Tocopherol LABELLING

Vitamin E is both lipophilic and non-toxic even at high doses so would be an excellent candidate as a lipophilic carrier for oligonucleotides. Therefore, as an addition to our cholesteryl product line, we offer simple a-tocopheryl (vitamin E) labelling. Totally synthetic a-tocopherol is racemic at its three chiral centers and is used to prepare this product.

psoralen LABELLING

Psoralen C2 at the 5’-terminus of an oligonucleotide serves effectively as a cross-linking reagent in double-stranded oligonucleotides. The 6 atom spacer arm of Psoralen C6 allows cross-linking with a triplex oligonucleotide strand.

Labelling with ultrafast Photo cross-linkers

When 3-cyanovinylcarbazole nucleoside (^CNVK) is incorporated into an oligonucleotide, very rapid photo cross-linking to the complementary strand can be induced at one wavelength and rapid reversal of the cross-link is possible at a second wavelength. Neither wavelength has the potential to cause significant DNA damage. Irradiation of a duplex containing a single incorporation of ^CNVK at 366nm led to 100% cross-linking to thymine base in 1 second, although complete cross-linking to cytosine takes 25 seconds.¹ A 30 second irradiation time should cover all situations. In addition, it was demonstrated that the purine bases were unreactive to cross-linking, allowing differentiation between pyrimidines and purines at the target site. The authors also determined the effect of sequence contexts around the ^CNVK site and demonstrated that the identity of bases on either side of the cross-linking site has little effect on the reaction. Once cross-linked, the UV melting temperature of the duplex was raised by around 30 °C relative to the duplex before irradiation. Complete reversal of the cross-link takes place at 312nm in 3 minutes. This facile reversal reaction is, therefore, accomplished with no damage to normal DNA.

In a later publication, a further application of this cross-linking technique was investigated.² When ^CNVK was cross-linked with a dC residue in duplex DNA, heating at 90°C for 3.5 hours led to deamination of the cytosine base to form uracil in the complementary strand. Reversal of the cross-link at 312nm led to a DNA strand in which dC had been converted to dU. The authors showed that this transformation is specific for the dC residue opposite the ^CNVK and any further adjacent dC residues are unaffected. Similarly, the authors have shown that ^CNVK can be cross-linked to an adjacent RNA strand.³

EDTA LABELLING

EDTA-C2-dT phosphoramidite contains the triethyl ester of EDTA which allows sequence-specific cleavage of single- and double-stranded DNA and RNA. The cleavage reaction is only initiated once Fe(II) and dithiothreitol are added and so is readily controlled. Coupling of EDTA-dT is normal but cleavage and deprotection should be carried out with sodium hydroxide in aqueous methanol (0.4M NaOH in methanol/water 4:1) overnight at room temperature.

Ferrocene Labelling

With an excellent stability profile, ferrocene has always attracted considerable interest for DNA labelling to generate probes for electrochemical detection. Based on our Amino-Modifier C6-dT structure, Ferrocene-dT is easily added to oligonucleotides with no disruption of regular hybridization behavior. Multiple incorporations into an oligonucleotide probe are also simply achieved. Oligonucleotides are deprotected using standard techniques. Ferrocene oligonucleotides should be stored under Argon and aqueous solutions should be degassed immediately.

LABELLING with Metal Chelates

2,2’-Dipicolylamine is a versatile metal-coordinating ligand capable of forming complexes with common metal ions including Zn2+, Ni2+, Cu2+, or Ag+. A tremendous advantage of dipicolylamine is complete compatibility with standard DNA synthesis, cleavage and purification protocols. Other chelating ligands may require nonstandard conditions or additional protection and deprotection steps. This product was manufactured and developed by Syntrix Biosystems Inc. Patents Pending. For Research Use Only.

Puromycin CPG

One of the most challenging requirements associated with combinatorial chemistry is the recovery of sequence information of the oligonucleotide or peptide selected by the screening assay. A method¹ has been developed to generate a fusion product between mRNA and the polypeptide it encodes using in vitro translation of synthetic RNAs 3’-labeled with puromycin, an antibiotic that mimics transfer RNA. Puromycin binds in the ribosome’s A site, forms a peptide bond with the growing peptide chain, and blocks further peptide elongation. By linking puromycin to mRNA, a peptide-RNA fusion product results from the translation of the message linking the encoding mRNA with its peptide product.

Labelling for Photo-Regulation of Oligonucleotides

Photo-control, the use of ultraviolet or visible light to control a reaction, has a number of advantages over other external stimuli:

• Light does not introduce contaminants into the reaction system,
• Excitation wavelength can be controlled through the design of the photo-responsive molecule, and
• It is now straightforward to control irradiation time and/or local excitation.

When a photo-responsive molecule is directly attached to DNA as a receptor, photo-regulation of the bioprocess regulated by that DNA molecule could, in principle, be achieved. Such photo-responsive DNA could also be used as a switch in a DNA-based nano-machine. Professor Hiroyuki Asanuma and his group at the department of Molecular Design and Engineering of the Graduate School of Engineering of the Nagoya University (Japan) have developed an efficient method to achieve this goal. They have attached azobenzene to DNA and made it photo-responsive^1,2. Azobenzene is a typical photo-responsive molecule that isomerizes from its planar trans-form to the non-planar cis-form after UV-light irradiation with a wavelength between 300 nm and 400 nm (lmax is around 330 nm). Interestingly, the system reverts from the cis-form to the trans-form after further irradiation with visible light (wavelength over 400 nm). This process is completely reversible, and the azobenzene group does not decompose or induce undesirable side reactions even on repeated trans-cis isomerization. By introducing azobenzenes into DNA through D-threoninol as a linker, Asanuma and co-workers succeeded in achieving photo-regulation of:

• Formation and dissociation of a DNA duplex^3,4 and
• Transcription by T7-RNA polymerase reaction^5,6,7.