Fluorescent Oligonucleotide Labeling Reagents

Introduction

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As the use of synthetic oligonucleotides in biomedical research gets more sophisticated, the simple modification of oligonucleotides becomes an urgent need. Although the automated oligonucleotide synthesis rapidly advances, there are still challenges in meeting the needs for increasingly larger quantities of modified oligonucleotides for therapeutic applications; better high-throughput methods for the screening and PCR markets; and improved synthesis quality of dye modified oligonucleotides for the diagnostic industry.

In recent years, dye-labeled oligonucleotides have received great attentions due to their important biological applications. For certain applications, such as DNA sequencing and in situ hybridization (e.g. FISH), oligonucleotides are usually required to be singly labeled. Subsequent detection and analysis relies on the fluorescent properties of the dye, most of which emit light in the visible spectrum. On the other hand, there are some types of biological applications, e.g. probes for real-time PCR quantification of DNA and RNA and allele discrimination (Molecular Beacons™), which require that oligonucleotides be doubly labeled. In the molecules of doubly labeled oligonucleotides one dye acts as a fluorophore, the other as a quencher. When dual-labeled probes are inactive, the light emission from the fluorophore must remain undetected and is absorbed by the quencher dye via a process of so called Fluorescent Resonance Energy Transfer (FRET). Because FRET is a distance-dependent interaction between the excited state of the donor and acceptor dye molecules, their eventual separation in the detection event allows the fluorescence to be detected.

To maximize the FRET efficiency, the FRET pairs need to be carefully selected based on the consideration of quite a few factors such as fluorescence lifetime, the spectral overlap of donor emission with acceptor excitation. AAT Bioquest offers the most comprehensive product line of FRET building blocks for labeling oligonucleotides, including both the classic dyes and our outstanding Tide Fluor™ (donors) and Tide Quenchers™ (acceptors).

5'-Labeling of Oligonucleotides Using Dye Phosphoramidites

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Because conventional automated synthesis proceeds from 3' to 5', the 5'-terminus is clearly a good choice for modifying oligonucleotides. As reported in a number of publications, the ability to attach a suitable molecule to the 5'-terminus for use as a label plays a critical role in the continuing development of non-radioactive probes and in DNA sequencing and amplification. A general approach to the modification of the 5'-terminus is to use reagents that would couple to the 5'-hydroxyl of an oligonucleotide.

Dye phosphoramidite reagents have been readily adapted for use in automated synthesizers with little or no modification to existing protocols. These reagents are well compatible with automated DNA synthesizers. In general, dye-labeled oligonucleotides can be deprotected at room temperature in concentrated ammonium hydroxide for a minimum of 24 hours, or shorter time that is appropriated for the protecting groups on the monomers being used. FAM, Dabcyl and Tide Quencher™ (TQ)-labeled oligos can be heated to 55°C in ammonium hydroxide for extended periods of time. However, TET, TF-3 and Cy-3 labeled oligos are less stable and survive only for a few hours at 55°C. HEX, TF-5 and Cy-5 labeled oligonucleotides must be deprotected at room temperature and the residual ammonia should be removed immediately after deprotection.

Fluorescein Labeling: AAT Bioquest offers fluorescein-based phosphoramidites that contains no 4, 4'- dimethoxytrityl (DMT) group and can be added only once at the 5'-terminus, thereby terminating synthesis. These fluorescein products include FAM, TET and HEX. FAM phosphoramidite is designed to produce the same fluorescein-type structure as previously prepared using fluorescein isothiocyanate (FITC). The TET and HEX phosphoramidites are designed to take advantage of the multicolor detection capability of modern DNA sequencers and genetic analyzers. For stronger fluorescence intensity, pH-insensitivity and high photostability, we highly recommend that you try our Tide Fluor™ 1 (TF-1) which has the spectral properties that are essentially identical to those of fluorescein.

Rhodamine Labeling: The light-absorbing properties of TAMRA, and spectral overlap with several commonly used fluorophores, including FAM, HEX, TET and JOE, make it useful as a quencher for the duallabeled oligo probes. However, its intrinsic fluorescence contributes to the background signal, potentially reducing the sensitivity of assays based on TAMRA. Despite these limitations, TAMRA has been used extensively in the design of probe-based assays, perhaps most notably for Real-Time PCR.

Oligonucleotides can be labeled with TAMRA using two distinct methodologies. Under standard deprotection conditions, TAMRA is not sufficiently stable. It degrades in the presence of ammonium hydroxide. If standard deprotection is required, the oligonucleotide is normally synthesized with an amino group at either the 3'-, or 5'-end and labeled with TAMRA post-synthetically using TAMRA, SE (see below). Oligonucleotides synthesized using UltraMILD monomers can also be labeled directly with TAMRA at the 3'-end using 3'-TAMRA CPG support. Subsequent deprotection of the oligo using potassium carbonate in methanol adequately removes the base protecting groups.

Tide Fluor™ Labeling: TF dyes are optimized to maximize FRET performance through enhancing donor fluorescence intensity. Although EDANS, FAM, TAMRA, ROX, Cy 3 and Cy5 have been widely used to develop a variety of FRET probes, there are still a few limitations for using these dyes. For example, the weak absorption and environment-sensitive fluorescence of EDANS have severely limited its sensitivity for developing nucleic acid detection probes. Compared to EDANS, fluorescein-based probes (such as FAM, HEX, JOE and TET) have stronger absorption and fluorescence. However the fluorescence of fluorescein-based probes is strongly dependent on pH. They only exhibit the strongest fluorescence at higher pH. This pH dependence makes the fluorescein-based fluorescent probes inconvenient for the assays that require low pH. In addition, most of fluorescein-based probes have quite low photostability, which limits their applications in fluorescence imaging. Among cyanine dyes, nonsulfonated Cy3 and Cy5 are widely used for developing a variety of nucleic acid probes, but they have quite low fluorescence quantum yield in aqueous media. The sulfonated Cy3 and Cy5 have improved fluorescence quantum yield than those of non-sulfonate cyanines. However, the sulfonated Cy3 and Cy5 are more difficult to use in the synthesis of fluorescent oligonucleotides due to the lack of the corresponding sulfonated cyanine phosphoramidites, and are quite cost-prohibitive.

To address these limitations, we have developed Tide Fluor™ donor dyes that are optimized as building blocks for developing FRET oligonucleotides and peptides for a variety of biological applications. Our Tide Fluor™ dyes (such as TF1, TF2, TF3, TF4 and TF5) have stronger fluorescence and higher photostability than the typical fluorophores such as fluoresceins, rhodamines and cyanines as described above. Our TF2 has essentially the same excitation and emission wavelengths to those of carboxyfluoresceins (FAM), making them readily available for the biological applications done with fluoresceins, but has an enhanced performance with our TF2 probes. TF2 has much stronger fluorescence at physiological conditions, and it is much more photostable than FAM probes. Moreover, compared to other fluorescent dyes alternative to fluoresceins and Cy dyes (such as Alexa Fluor™ and Cy3, Cy5 and Cy7), Tide Fluor™ dyes are much more cost-effective with comparable or even better performance for some biological applications.

Key Features of Tide Fluor™ Donors

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• Optimized to pair with Tide Quencher™ dark acceptors to maximize the FRET potentials
• Stronger fluorescence intensity to enhance assay sensitivity
• pH-insensitive and environment-insensitive fluorescence to simplify assays
• Higher photostability to improve the quality of fluorescence imaging
• A variety of reactive forms available for conjugations

1. *Texas Red^® is the trademark of Molecular Probes, Inc.

Tide Quencher™ Acceptor Dyes: TQ dyes are optimized to maximize FRET performance through enhancing quenching efficiency. Although DABCYL has been used to develop a variety of FRET applications, its low quenching efficiency of longer wavelength dyes (such as fluoresceins, rhodamines and cyanines) have limited its use in the development of sensitive fluorogenic FRET probes. Additionally, the absorption spectrum of DABCYL is environment-sensitive. AAT Bioquest has developed the robust Tide Quencher™ acceptor dyes for the development of longer wavelength FRET probes. These Tide Quencher™ dark FRET acceptors (such as TQ1, TQ2, TQ3 and TQ4) are optimized to pair with our Tide Fluor™ dyes and the classic fluorophores (such as AMCA, EDANS, FAM, TAMRA, HEX, JOE, TET, ROX, Cy3, Cy5 and Cy7). Like our Tide Fluor™ donors dyes, our Tide Quencher™ acceptor dyes are much more cost-effective with comparable or even better performance for your desired biological applications than other similar products on the market.

We offer a variety of reactive forms for both our Tide Fluor™ donors and Tide Quencher™ acceptors. For in-synthesis labeling of oligonucleotides, we offer both phosphoramidites of our Tide Fluor™ and Tide Quencher™ dyes and their CPG supports. For post labeling of oligonucleotides, we offer both amino-reactive and thiol-reactive Tide Fluor™ and Tide Quencher™ dyes that are water-soluble. Our Tide Quencher™ dyes have been used for developing a variety of Molecular Beacon oligonucleotide probes. Tide Quencher™ dyes are great choice for you to eliminate the limitations of classic quenchers. Tide Quencher ™ dyes are excellent dark quenchers that are individually optimized to pair with all of the popular fluorescent dyes such as fluoresceins and rhodamines. Our Tide Quencher™ series of nonfluorescent dyes cover the full visible spectrum with unusually high efficiency. TQ2 has absorption maximum perfectly matching the emission of FAM while TQ3 is proven to be the best quencher for Cy3. In summary, our Tide Quencher™ dyes have the following advantages:

• Most Powerful: enable you to explore the FRET potentials that might be impossible with other quenchers.
• Versatile Reactive Forms: convenient for self-constructing your desired FRET biomolecules.
• Complete Set of Dyes: perfectly match your desired fluorescent donors.
• Enhanced Value: competitive price with the best performance.

1. *Texas Red^® is the trademark of Molecular Probes, Inc.

3'-Labeling of Oligonucleotides Using Dye CPG Supports

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Besides the dye CE phosphoramidites described above, AAT Bioquest also offers dye CPG supports. Dye CPG supports have traditionally been used to add the dye labels at the 3'-terminus. Dye CPGs are used to introduce a dye molecule to the 3'-terminus of oligonucleotides. Our dye CPGs are derived from dye carboxylic acids and are attached via an amide linkage, giving an oligo product that is much easier to be purified by HPLC. The use of dye CPGs in oligonucleotide synthesis proceeds in a manner analogous to the use of a normal nucleoside support with some necessary modifications. Different dye CPGs might require different cleavage methods. The cleavage of the oligonucleotides from the FAM and Tide Quenchers™ (TQs) supports is similar to the standard ammonium hydroxide cleavage. TAMRA CPG has to be deprotected under very mild conditions to safeguard the base-labile TAMRA fluorophore. We recommend the use of UltraMild monomers and the use of potassium carbonate in methanol for deprotection. An alternative procedure using t-butylamine/methanol/water (1:1:2) might allow the use of regular monomers.

Indirect Modification of Oligonucleotides

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AAT Bioquest currently offers 5'-amino-modifiers (Cat. # 4300 and Cat. # 4304). These reagents are designed for use in automated synthesizers to functionalize the 5'-terminus of a target oligonucleotide with a primary amine moiety. The resulted amino-modified oligonucleotides can be conjugated to a variety of tag molecules such as fluorophores, biotins, alkaline phosphatase and HRP. Due to the increased possibility of side reactions during the deprotection of modified oligonucleotides, it is recommended that the ammonium hydroxide treatment be carried out at a lower temperature than that used for unmodified oligonucleotides. The MMT protecting group of the 5'-amino-modifier (Cat. # 4300) can be removed on the synthesizer by deblocking until the yellow color elutes totally. The solution of MMT cation produced by acid deprotection is yellow and is not well quantified by trityl monitors. The modified oligonucleotide may be purified using a Poly-Pak cartridge, HPLC or gel electrophoresis. Poly-Pak cartridge purification is accomplished using the trityl-on procedure. HPLC may be performed either before or after the attachment of the label. If purification is desired prior to the label attachment, the MMT group should not be removed from the oligonucleotide as the lipophilic character of the MMT group aids in HPLC purification.

AAT Bioquest also offers Chemical Phosphorylation Reagent (CPR, Cat. # 6001). CPR has proved to be fast and convenient for chemical phosphorylation of the 5'-terminus of oligonucleotides. In addition, this reagent has proved its utility for simple phosphorylation of the 3'-terminus. It is introduced as the first addition to any nucleoside support, followed by normal synthesis of the target oligonucleotide. After the standard ammonium hydroxide deprotection, the linkage decomposes and is ß-eliminated from the target molecule, leaving a phosphate group at the 3'-terminus. The final DMT group may be removed on the synthesizer or it may be retained to aid in purification. If the DMT group is retained, it may be removed on a purification cartridge or, by treating the oligonucleotide with acetic acid:water (80:20) at room temperature for 1 hour following purification.

Post-Labeling of Oligonucleotides Using Dye Succinimidyl Esters

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Succinimidyl esters are proven to be the best reagents for labeling amine-modified oligos because the amide bonds formed are essentially identical to, and as stable as the natural peptide bonds. These reagents are generally stable and show good reactivity and selectivity with aliphatic amines. AAT Bioquest offers highly purified classic fluorescent dye succinimidyl esters and our outstanding Tide Fluor™ (TF) and Tide Quencher™ (TQ) succinimidyl esters. Our dye succinimidyl esters are packed under nitrogen to enhance their shelf life, and also packed in different sizes to provide you the maximum convenience in handling these moisture-sensitive reagents.

There are a few factors that need to be considered when SE compounds are used for conjugation reactions:

Solvents: For the most part, reactive dyes are hydrophobic molecules and should be dissolved in anhydrous dimethylformamide (DMF) or dimethylsulfoxide (DMSO).

Reaction pH: The labeling reactions of aliphatic amine-containing oligonucleotides with succinimidyl esters are strongly pH dependent. Amine-reactive reagents react with non-protonated aliphatic amine groups, including the amine groups of oligos. Thus amine acylation reactions are usually carried out above pH 7.5. Oligo modifications by succinimidyl esters can typically be done at pH ranging from 7.5 to 8.5, whereas isothiocyanates may require a pH 9.0-10.0 for optimal conjugations.

Reaction Buffers: Buffers that contain free amines such as Tris and glycine and thiol compounds must be avoided when using an amine-reactive reagent. Ammonium salts (such as ammonium sulfate and ammonium acetate) must also be removed (such as via dialysis) before performing dye conjugations.

Reaction Temperature: Most conjugations are done at room temperature. However, either elevated or reduced temperature may be required for a particular labeling reaction.

Why should you use our Tide Quencher™ dyes (TQ1, TQ2, TQ3, TQ4 and TQ5)?

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• Most Powerful: TQs enable you to explore the maximum FRET potentials with oligonucleotides;
• Versatile Reactive Forms: TQs are convenient for self-constructing your desired FRET biomolecules;
• A Complete Set of Dyes: TQs perfectly match any fluorescent donors that you select;
• Enhanced Value: We offer you the most competitive price with the best performance.

Why should you use our Tide Fluor™ dyes (TF1, TF2, TF3, TF4 and TF5)?

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• Optimized to pair with Tide Quencher™ dark acceptors to maximize the FRET potentials
• Stronger fluorescence intensity to enhance assay sensitivity
• pH-insensitive and environment-insensitive fluorescence to simplify assays
• Higher photostability to improve the quality of fluorescence imaging
• A variety of reactive forms available for conjugations