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3'-Cyanine 5 CPG Column

3'-Cyanine 5 CPG Column

CPG column used to incorporate the cyanine-5 dye moiety onto the 3'-end of an oligonucleotide.
  • Used to add a red fluorescent dye to the 3' end of an oligonucleotide.
  • Maximal emission around 667 nm. Alternative to Quasar 670.
  • Quenched by BHQ-2.
  • 1000 Å CPG suitable for highly modified oligonucleotides (> 20mers).
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Product information

Cyanine-based dyes have been used for many years in areas such as textiles. Their application in nucleic acid labelling (1) increased firstly with their commercial availability as succinimidyl esters, then latterly as phosphoramidites. Today they are a central part of many diagnostic platforms and assays based on fluorophore labelling and detection.

Cyanine dyes are used as fluorescent markers in oligonucleotide synthesis,(2) primarily for molecular diagnostics such as the preparation of probes used in monitoring real-time PCR, fluorescence in situ hybridisation (FISH) and in Surface-Enhanced Resonance Raman Spectroscopy (SERRS) based DNA detection assays. Their emission spectra can be tuned by altering the length of the polymethine chain and solubility in organic or aqueous solvents can be altered via the substituents on the aromatic ring.

The most commonly used classic phosphoramidite dyes are Cyanine-3 and Cyanine-5 (structurally equivalent to Cy™ 3 and Cy™ 5. These are generally attached to the 5'-end of an oligonucleotide during synthesis, however are easily incorporated within the sequence. The MMT-protected hydroxyl group is removed in the same way as DMTr protection. Internal incorporation is not common due to the lack of heterocyclic base in their structure and as such they do not have the ability to participate in base pairing. This destabilises any duplexes formed.

For 3'-attachment, we have introduced the equivalent 3'-modified CPG supports. Previously this was done by adding the dye post- synthetically onto an amino-modified oligonucleotide or by adding the amidite to a support functionalised with a modification that will not interfere with the use of the oligonucleotide (e.g. phosphate, spacer). 3'-Labelling is particularly useful in FRET where the FRET partner is incorporated either at the 5'-end or within the oligonucleotide sequence.(3)

LGC Biosearch Technologies' series of Quasar™ dyes were developed as direct replacements for the Cy™ 3, Cy™ 5 and Cy™ 5.5 dyes; Quasar 570, Quasar 670 and Quasar 705, with emission maxima at 570, 670 and 705 nm, respectively. Quasar dyes increase fluorophore choices and help eliminate potential cross-talk problems associated with multiplex qPCR. They are efficiently quenched by BHQ-2 or BHQ-3, and can be used in many different probe formats. Multiplex suggestions and PCR instrument compatibility of Quasar dyes can be found at our multiplex qPCR webpage.

Quasar CPG and phosphoramidites are available for direct labelling of oligonucleotides. Active esters are available for coupling to amine groups on substrates such as proteins, peptides and oligonucleotides. The carboxy acid variants are used to label peptides, oligonucleotides or functionalized amines.

Ref:

  1. Molecular Probes Based on Cyanine Dyes for Nucleic Acid Research, T.G. Deligeorgiev, p125, in Near Infrared Dyes for High Technology Applications, Ed. S. Daehne, U. Resch-Genger and O. Wolfbeis, NATO ASI Series Publ., Kluwer Academic, 1998.
  2. Fluorescence based strategies for genetic analysis, R.T. Ranasinghe and T. Brown, Chem. Commun., 5487- 5502, 2005.
  3. Fluorescence resonance energy transfer in near-infrared fluorescent oligonucleotide probes for detecting protein-DNA interactions, S. Zhang, V. Metelev, D. Tabatadze, P.C. Zamecnik, A. Bogdanov Jr, Proc. Natl. Acad. Sci. USA, 105, 4156-4161, 2008.

Applicable Products

LK2158 Quasar™ 570-CE Phosphoramidite
LK2159 Quasar™ 670-CE Phosphoramidite
LK2412 3'-Cyanine-3 SynBase™ CPG 1000/110 (Cyanine 540)
LK2413 3'-Cyanine-5 SynBase™ CPG 1000/110 (Cyanine 650)
LK2520 Cyanine-3-CE Phosphoramidite (Cyanine 540)
LK2521 Cyanine-5-CE Phosphoramidite (Cyanine 650)

Physical & Dilution Data

Dilution volumes (in ml) are for 0.1M solutions in dry acetonitrile (LK4050) with the exception of LK2521 where anhydrous alcohol free DCM is required. Adjust accordingly for other concentrations. For µmol pack sizes, products should be diluted as 100µmol/ml to achieve 0.1M, regardless of molecular weight. We suggest also using 3-4Å activated molecular sieves (10% v/v).

Item

Mol. Formula

Mol. Wt.

Unit Wt.

250mg

500mg

1g

LK2158 C44H65N5O4P 759.00 622.74 3.29 6.59 13.18
LK2159 C46H67N5O4P 758.04 648.78 3.18 6.37 12.74
LK2412 - - 507.59 - - -
LK2413 - - 633.63 - - -
LK2520 C58H70ClN4O4P 953.64 507.59 2.62 5.24 10.49
LK2521 C60H72ClN4O4P 979.68 533.63 2.55 5.10 10.21

Coupling

A 10min coupling time is recommended for all phosphoramidites. Due to the milder conditions required when deprotecting cyanine-labelled oligonucleotides, the use of fast or ultramild deprotection amidites (Ac-dC, Bz-dA, dmf-dG, iPrPac-dG, Pac-dA) is recommended. The use of 4A molecular seives is recommended.

Deprotection

As these reagents require no deprotection themselves, the oligonucleotide deprotection strategy employed is guided by the protective groups on the rest of the oligo. However, the mildest possible conditions are recommended, especially for Quasar™ 670 (LK2159) and Cyanine 5 (LK2521) which are susceptible to degradation at elevated temperatures.

Cleavage and deprotection: Do this in one step. Remove the resin from the column and place into a deprotection tube. Add ammonium hydroxide and allow to stand for 2-3hr at room temperature. The deprotection solution must be removed immediately after deprotection (see below).

Biosearch recommend deprotection of the Quasar dyes with ammonium hydroxide for 8hr at 25°C or for 1 hr at 60°C, however we have obtained good quality oligos with the above conditions, especially as elevated temperatures increase degradation.

Removing the deprotection solution: This must be done as soon as the deprotection step is complete. It is important not to heat the oligo to remove the deprotection solution since this will degrade the dye. Pass the solution through a G25 (NAP10 or equivalent) column. This will remove the basic solution and the sample is ready for purification. Some users pass the solution through two columns to be absolutely sure that the solution is now ~ neutral pH.

Spectral Data

 

Absorbance Max./nm

Emission Max./nm

Colour

Quasar™ 570 547 570 Yellow-Orange
Quasar™ 670 644 670 Red
Cyanine 3 546 563 Yellow-Red
Cyanine 5 646 662 Violet-Far Red

Storage & Stability

These products are light-sensitive and are susceptible to oxidation when left exposed to air and/or moisture. Store dry in a freezer, in a sealed dark or amber container at –20°C. Similarly, modified oligonucleotides must also be protected from exposure to light. The phosphoramidites are stable in solution under argon for 24h.

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