Modifier Descriptions

Tags & Modifiers

Acridine:   Acridine is an effective intercalating agent as well as being a lipophilic carrier molecule. Acridine labeled oligonucleotides are sometimes used in antisense research. Oligos containing acridine cannot be HPLC purified, nor can they be analyzed by HPLC, as they bind irreversibly to the column. Acridine is available as both a 3' and a 5' modifier. Labeling is performed during synthesis and is not a benchtop reaction.

Amino Modifiers: Incorporation of amine modifiers allows for specifically targeting labeling and cross-linking reactions to a defined site in an oligo. These primary amines provide reactive sites for a variety of purposes including immobilization of oligos on solid supports, crosslinking of oligos to a second molecule, and benchtop labeling of the oligo. A variety of amino modifiers are available. They can be used to label oligos at the 5' end, the 3' end, and internally. The length of the linker arm, connecting the amine group and the base, may affect the efficiency of reactions involving the amine. We generally recommend the C6 linker for post-synthesis modification of oligos with the amino group. Amino dT is the modifier used to label oligos internally and is the reagent of choice for addition of multiple amine residues within the same oligo. The 3' amine modifiers introduce a primary amine group at the 3' terminus of the oligo. Because oligos are synthesized from a 3' to 5' direction, all molecules will be modified with the 3' amino group. The 3' modifiers are usually used to end label or immobilize oligos. Again, we generally recommend a C6 linker arm on the amino group for most applications. Building off a modified solid support may reduce the quality and yield of an oligo and is a particular problem for oligos longer than 35mers. The modified solid support is not designed for synthesis of oligos longer than a 35mer, therefore you should consider a 5' modifier or internal modifier on oligos longer than 35mers. Use of an internal modifier can alleviate this problem. Like the 3' amine modifiers, the 5' amine modifiers introduce a primary amine group, except at the 5' terminus of the oligo. As a consequence of the chemistry, only full-length oligos will be labeled at the 5' terminus. We recommend a 5' unilink with a C6 linker arm for most purposes. The unilink has a group that allows measurement of the coupling efficiency of the reaction that adds the 5' amine to the oligo. All of the amine labeling reactions are performed during synthesis on the machine and are not benchtop reactions.

Biotin Labeling: Incorporation of biotin modifiers provides a means to specifically detect a labeled oligo. Biotin labels are increasingly used in nonradioactive detection technologies, for example, chemiluminescent, visible dye, and fluorescent detections. These biotin modifiers provide sites for streptavidin or avidin binding to the oligo. Streptavidin or avidin binding can be used to amplify the detection signal from the biotin-labeled oligo. Biotin modifiers can be used at the 5' terminus, the 3' terminus, and internally. Biotin-dT and Biotin-dC are the reagents normally used to label an oligo at internal positions. The linker arm for the biotin dT is C16, which decreases steric hindrance when using multiple biotins. This modifier can be used alone, or in combination with other internal, 5', or 3' biotin modifiers to introduce multiple biotin groups. Since oligos are synthesized from 3' to 5', all molecules labeled with a 3' biotin are modified. These modifiers efficiently block enzyme-mediated extension from the 3' terminus of the oligo. We recommend 3' Bio-TEG modifier for the majority of experimental applications that require a 3' biotin. Other 3' biotin modifiers are available on request. The building of a modified solid support (the 3' end) may reduce the quality and yield of an oligo and is a particular problem for oligos longer than 35mers. The modifier solid support is not designed for synthesis of oligos longer than a 35mer, therefore you should consider a 5' modifier or internal modifier on oligos longer than 35 bases. As a consequence of the synthesis chemistry, the 5' biotin modifiers only label full-length oligos at the 5' end. We recommend 5' Biotin-ON for most purposes that call for a 5' biotin. The Biotin-ON structure has a group that allows coupling efficiency to be easily monitored. This monitoring results in a more consistent product. Other 5' biotin modifiers are available on request. If needed for specific experimental applications, linker arm length can be increased by the use of spacers. Regardless of the biotin label chosen. All of the biotin labeling occurs during the course of synthesis on the machine and is not a benchtop reaction.

Branching Modifiers: Branching modifiers directly incorporate a branching site into an oligo during the synthetic process. There are two options for branching, a symmetric branch and an asymmetric branch. Incorporation of the symmetric branch creates two identical branches, and chain elongation occurs from both branching sites simultaneously. The asymmetric branching modifier creates two different branches, and chain elongation is initiated separately on each arm to result in an asymmetrically branched oligo.

5-Bromo-deoxy Cytidine: 5-Bromo-deoxy Cytidine is a halogenated pyrimidine analog. It can be used for photo-crosslinking due to its photolability. It is also used in crystallographic studies.

5-Bromo-deoxy Uridine: 5-Bromo-deoxy Uridine is a halogenated pyrimidine analog. It can be used for photo-crosslinking due to its photolability. It is also used in crystallographic studies.

Carboxy-dT: Carboxy-dT can be used in place of a dT in an internal position of the oligo, or as the 5' terminal base. The carboxy-dT modifier allows direct coupling of an oligo to a molecule or surface with a primary amine. This modifier is added to the oligo on the machine during synthesis.

Cholesterol and Cholesteryl: Cholesterol and cholesteryl modifiers are used to facilitate cell entry of modified oligos. Cholesterol-modified oligos can be used in conjunction with lipofectin, or liposome incorporation of the oligo. The presence of these modifiers does not affect the duplex melting temperature of the oligo/target hybrid. It is not known if cholesterol-modified oligos have any side effects on cellular function. The cholesterol type modifications are available to tag either the 5' or 3' end of the oligo. Cholesterol is the form that is available for labeling the 3' end of oligos. Incorporation of the label is by means of the solid support during synthesis on the machine. Therefore, from a synthetic standpoint, all oligos in the synthesis will be labeled with the 3' modifier. Cholesteryl is the form of the modifier available for labeling the 5' end of the oligo. As with the 3' modifier, labeling occurs during the course of synthesis on the machine. The 5' cholesteryl modification results in a mixture of modified and unmodified oligos. As a consequence, two bands are generally visible on a gel photo after 5' labeling. One band represents a full-length unmodified oligo, and the other band represents a full-length modified oligo. In general, cholesterol-modified oligos can be difficult to dissolve in aqueous solutions. In most cases a freeze/thaw cycle may help to get the oligo into solution. We recommend keeping cholesterol-modified oligos in solution.

Cordecypin: Cordecypin is an alternative name for 3' deoxy A.

3'-Deoxy Terminator Bases: The 3'-deoxy bases can be used as terminators at the 3' terminus of an oligo. They are excellent blocking groups, preventing polymerase extension from the 3' terminus. These bases can also be used internally to create a 5'-2' phosphate linkage in the oligo. All four bases are available with the deoxyribose sugar blocked at the 3' position, to give 3'-deoxy terminator G, T, C, and A. Cordecypin is an alternative name for the 3'-deoxy A terminator. These modifiers are preferred in place of dideoxy modifications.

7-Deaza-deoxy Adenosine: This modifier is used to study the effect on an oligo when critical groups for hydrogen bonding are modified. 7-deaza dA lacks the ability to form as many hydrogen bonds as dA. It is therefore useful for studying the structural effects of this modification.

7-Deaza-deoxy Guanosine: This modifier is used to study the effect on an oligo when critical groups for hydrogen bonding are modified. 7-deaza dG lacks the ability to form as many hydrogen bonds as dG. It is therefore useful for studying the structural effects of this modification.

2,6-Diaminopurine 2'-deoxyribose: This purine analog for dA is capable for forming an additional hydrogen bond with dT. It is useful for studies involving duplex stabilization.

3'-Dideoxy Terminator Bases: Dideoxy terminator bases to prevent extension from the 3' terminus have been replaced by the 3'-deoxy terminator bases described above. Synthesis efficiencies for 3'-dideoxy terminators are poor. We suggest using 3'-deoxy Terminators as a substitute.

Digoxigenin: Digoxigenin can be attached to oligos in a benchtop labeling reaction. The oligonucleotide may be modified in one of two types of reactions. In the first type of reaction the oligocucleotide is "tailed" using terminal deoxynucleotidyl transferase and dig-11-dUTP. Multiple labels are attached at the 3' end of the oligonucleotide but there may be a loss of specificity due to homology of the "tail" to unwanted sequences, e.g., poly-A RNA. Oligos Etc. provides the second type of labeling in which the oligonucleotides are modified during synthesis to contain amino groups. The amino-modified oligonucleotides are labeled with digoxigenin-NHS-ester. The positions of amino-modification can be specified within an oligo. The 5' terminus can be labeled with a C6 or C12 linker. C12 may be preferable. Linker arm length determines accessibility of the digoxigenin label to the detecting antibody. Amino-dT can be substituted for dT at internal positions of the oligo. The spacing between labels should be maintained at 10 or more bases to prevent steric limitations on antibody recognition of the digoxigenin label. Multiple labels are necessary for northern and southern hybridization probes. The digoxigenin label is destroyed by even brief exposure to ammonium ions, for example during ethanol precipitation.

Dyes: Many different dye labels are possible for oligonucleotides. See the dyes section for a listing of possible dye labels. Dyes are commonly used for detection of oligonucleotides or DNA. Dye labeling has become very common. We offer different methods of purification for dye labeled oligonucleotides depending on the type of dye. For example, not all dyes are stable under PAGE conditions. Some cannot be assayed by ion-exchange HPLC without showing evidence of degradation due to the analysis technique. Specific recommendations are available from our technical services group.

Etheno dA: Etheno dA is an analog of deoxy A. Etheno dA is a fluorescent molecule, which is useful in studying the transition between DNA structures. Due to its structure, etheno dA will not base pair with dT or dU. Therefore, oligos with etheno dA in the middle or at the 3' end cannot serve as PCR or sequencing primers. If the etheno dA is located at the 5' end, the oligo can still serve as a primer.

5-Fluoro-deoxy Uridine: Fluoro-deoxy uridine is a base analog that has the potential to bind to A and G. It does not destabilize duplex formation, and it is an alternative to using the mixed bases A/G for degeneracy.

Inosine Modifiers: Deoxy inosine is a base analog that has the potential to bind to A, C, G, or T. However, there have been some reports that there is a preference for binding to C. Deoxy inosine does not disturb DNA duplex formation and does not destabilize the duplex. It has been reported that deoxy inosine containing primers work better than primers containing mismatches at the same positions. Both ribo inosine and deoxy inosine modifiers couple with lower efficiencies than the corresponding deoxy or ribo A,C,G,T bases.

5-Iodo-deoxy Cytidine: 5-Iodo dC is a photoactive analog of dC. It is useful for crosslinking studies. We suggest handling under low light conditions.

5-Iodo-deoxy Uridine: 5-Iodo dU is a photoactive analog of dT. It is useful for crosslinking studies. We suggest handling under low light condtions.

Inverted Bases: Inverted bases are utilized primarily for antisense purposes. The natural 3'-5' linkage is modified to the 3'-3' linkage and/or a 5'-5' linkage. This modification from the natural linkage confers added protection against nuclease activity, which is an area of concern, particularly in antisense experiments. Inverted bases may also be used for 5' to 3' synthesis in those rare instances where only a 5' modifier is available but it must be appended to the 3' terminus.

5-Methyl-deoxy Cytidine: 5-methyl dC is a pyrimidine analog of dC. It is used to enhance base pairing with dC. The increase in hybridization efficiency is due to the hydrophobic nature of the methyl group at the C-5 position, which helps to exclude water molecules from the duplex.

N-6-Methyl-2' deoxy Adenosine: Methylated modifiers are used to study the mutagenic effects that occur as a result of methylation of exocyclic amines in DNA.

O-4-Methyle-deoxy Thymidine: Methylated modifiers are used to study the mutagenic effects that occur as a result of methylation of exocyclic amines in DNA.

O-6-Methyl-deoxy Guanosine: Methylated modifiers are used to study the mutagenic effect that occur as a result of methylation of exocyclic amines in DNA. Oligos containing this modifier require a special deprotection protocol. The deprotection is involved. We can supply the oligo still on the solid support to be deprotected by our customers, or we can complete the deprotection here.

5-O-Methyl-deoxy Thymidine: Methylated modifiers are used to study the mutagenic effect that occur as a result of methylation of exocyclic amines in DNA.

8-Oxo-dG: This type of DNA modification occurs naturally by oxidation and ionizing radiation. Therefore, 8-oxo-dG is used to study the mutagenic effects of this modification.

2'-deoxy Nebularine: Like deoxy inosine, deoxy nebularine is a base analog that has the potential to bind to A, C, G, or T. However, similar to inosine, there have been some reports that there is unequal binding to the four bases.

Phosphate Modifiers: The use of the 3' phosphate modifier, or the 5' phosphate modifier, eliminates the need for enzymatic phosphorylation of oligos before ligation or any other protocol that requires a 3' or 5' phosphate on the oligo. The addition of the phosphate label occurs during the course of synthesis on the machine. As a result, the efficiency of phosphorylation is much greater than that of the corresponding enzymatic reaction. The addition of a phosphate to either or both ends of an oligo will change the mobility of the oligo on a gel. A phosphorylated oligo runs faster (higher mobility) in a denaturing gel than the same sequence with hydroxyl group(s) in place of the phosphate(s).

Psoralen: Psoralen is an intercalating agent that forms photoadducts with thymine residues when exposed to longwave UV. Psoralen is available for 5' oligo labeling. The labeling is performed during synthesis on the machine. HPLC purification cannot be performed on oligos that contain this modifier.

Puromycin: Puromycin is an antibiotic that mimics transfer RNA. Puromycin binds in the ribosome's A site and forms a peptide bond with the growing peptide chain to block peptide elongation. By linking puromycin to synthetic RNA, a peptide-RNA fusion product can be formed.

Spacers: Spacer molecules are used to bridge sections of oligos where no binding is possible and to space modifiers further from the oligo. The spacers can be added to substitute for unknown bases in a sequence. The dSpacer allows for incoroporation of a blank (abasic) site in the oligo while maintaining the correct spacing along the sugar phosphate backbone for each of the nucleotides in the sequence. The dSpacer can be used at the 3' end, 5' end, or internally. Spacer 3, 9, and 18 can all be added to substitute for unknown bases in a sequence, as well as to create distances of various lengths between the oligo and a modifier. Spacer C12 adds a very hydrophobic region to an oligo.

6-Thiol-deoxy Guanine: 6-Thiol-dG has been used to treat human malignancies. This modifier is most likely converted to the nucleoside 6-thioguanisine and causes cytotoxic effects because of the damage caused by strand cleavage or cross-linking to DNA or proteins.

Thiol Modifiers:   There are two thiol modifiers available for coupling to an oligo, a C6-disulfide (C6 S-S) and a C6-thiol modifier (C6 S-H). The C6-thiol modifier incorporates a protected free thiol group in an oligo. After deprotection a free thiol group is available for attaching reagents such as horseradish peroxidase or alkaline phosphatase to an oligo. The C6-thiol modifier (C6 S-H) must be used immediately after deprotection to ensure maximum coupling efficiency to a target and to prevent oxidative dimerization of the oligo. We recommend that the user perform the final deblock to maintain the integrity of the product. The oligo is shipped as a lyophilized pellet along with the deprotection protocol for deblocking the thiol linker. The C6-disulfide (C6 S-S) incorporates a disulfide functional group on the oligo. The disulfide group is stable to the ammonium hydroxide used in the deprotection process but is easily cleaved to DTT to produce a free thiol group. As with the C6-thiol modifier, this free thiol group can be reacted with maleiimide to iodoacetyl conjugates. In addition, the C6-disulfide can be used to generate a linker that can subsequently be cleaved by DTT (i.e., if a biotin miodifier is added to the 5' end of an oligo after the addition of a 5' thio-modifier C6- S-S, then DTT can be added to cleave the oligo from the biotin, for instance after reacting with streptavidin).

Internal Thiol Modifiers: There are three modifiers that can be used to introduce a thiol group internal to the oligonucleotide. They are 6-Thio-dG, 4-Thio-dT , and 4-Thio-dU. Each of these modifiers will introduce an available thiol group that can be used for crosslinking.

Deoxy-Uridine: Deoxy-uridine is incorporated in place of deoxyT under circumstances where it is desirable to chew up one of the two strands of DNA. A specific enzyme is utilized that chews up DNA that has deoxyU incorporated in the backbone.

Universal Bases: This type of modifier can base pair with all four of the standard bases. Designing primers from protein sequences by backtranslation usually results in degenerate oligonucleotides. Universal bases are used to reduce oligonucleotide degeneracy. The best known of these types of modified bases are 2'-DeoxyInosine and 2'-DeoxyNebularine. Neither of these two bases binds to all four bases evenly, but they are a frequently used method of reducing degeneracy. As an alternative, bases such as 3-Nitropyrrole 2'-deoxynucleoside and 5-Nitroindole 2'-deoxynucleoside can be used to take advantage of duplex stabilization by base stacking effects. The degenerate bases dP and dK can form base pairs with purines and pyrimidines, respectively, and can also be used to reduce the overall degeneracy of an oligonucleotide.