Affinity Ligands Oligonucleotide Synthesis

Click here for a list of other Affinity Ligand Modifications.

Biotin is an affinity label that can be incorporated at either the 5'- or 3'-end of an oligonucleotide, or at an internal position using biotin dT or Amino bases for conjugation to biotin-NHS. Biotin has a high affinity for the bacterial protein, streptavidin, which can be conjugated to a solid support (such as magnetic beads) for use as a capture and immobilization medium for a biotinylated oligo. In the biotin phosphoramidite, the biotin is attached to a long spacer arm, which acts to minimize steric hindrance between the biotin moiety and the oligo, thereby providing streptavidin easy access to the biotin. Biotinylated oligos are most commonly used as probes or primers in a variety of in vitro and in vivo applications.

Besides their importance as nucleic acid probes, biotinylated oligonucleotides are also useful for the purification of DNA binding proteins. In this context, the biotinylated oligonucleotide can be bound to a streptavidin matrix and used for either column or spin chromatography. For isolation of DNA binding proteins, the streptavidin-biotin-oligonucleotide complex is incubated with a crude cell extract containing nuclear proteins. Following appropriate washes, the proteins that bind selectively to the oligonucleotide sequence can be eluted under conditions that disrupt the protein:DNA complex. Because the binding of biotin to streptavidin is essentially irreversible and is resistant to chaotropic agents and extremes of pH and ionic strength, the elution conditions can be relatively stringent.

Dual Biotin

Dual Biotin modification specifically can be used to add multiple biotin moieties at the 5'- or 3'-end of an oligo. The most common use of this modification is to incorporate two biotin (Dual Biotin) molecules in sequence (separated by a six-carbon linker) at the 5'-end of an oligo. This "Dual Biotin" has higher binding affinity for streptavidin than that of a single biotin. The additional binding strength can be critical for applications requiring the use of biotinylated DNA attached to streptavidin-coated beads at higher temperature (for example, in PCR). Dual Biotin is known to prevent or effectively reduce loss of biotinylated DNA from such beads during heating (1). Dual biotin also is used to label the linker primers in Serial Analysis of Gene Expression (SAGE) protocols (2).

For direct biotin-labeling of target RNA transcripts for microarray analysis, a special 3'-biotinylated donor nucleotide molecule containing three biotin molecules in sequence was synthesized and then ligated to the target RNA using T4 RNA ligase. The attachment of three biotins to RNA in this manner resulted in a 30% increase in target signal intensity and improved transcript detection sensitivity (3)

.

Multi Biotin

Multi-Biotin is a modification that can be added sequentially as many units as desired based on the application. Use Dual Biotin modification code [Bio-Dual] that is specific for two units of biotin.

For direct biotin-labeling of target RNA transcripts for microarray analysis, a special 3'-biotinylated donor nucleotide molecule containing three biotin molecules in sequence was synthesized and then ligated to the target RNA using T4 RNA ligase. The attachment of three biotins to RNA in this manner resulted in a 30% increase in target signal intensity and improved transcript detection sensitivity (3)

.

Desthiobiotin

Desthiobiotin is a biotin derivative. Like biotin, desthiobiotin binds to streptavidin, but its binding affinity is considerably less (2x10E-9 M) than that of biotin (4.0x10E-14 M) (1). Consequently, oligonucleotides labeled with desthiobiotin can be easily displaced from streptavidin by biotin, thereby making recovery of the labeled oligo (for example, in affinity purification protocols) from a streptavidin-coated support a relatively simple process (2). Desthiobiotin-labeled oligos can also be conveniently eluted from streptavidin-coated supports by incubation in distilled water at 95C for 10 minutes (3). Gene Link recommends substitution of of desthiobiotin for biotin for those cases where reversible capture of oligonucleotides is desirable. Note that since desthiobiotin is in the form of an NHS ester, an active primary amino group (such as Amino Linker C6) must first be incorporated into the oligonucleotide, to allow for subsequent conjugation to desthiobiotin NHS ester.

Desthiobiotin NHS modification is a post synthesis conjugation to a primary amino group thus an additional modification with an amino group is required. A C6 or C12 amino group can be placed at the 5' or for the 3' end a C3 or C7 amino and for internal positions an amino modified base is used, e.g Amino dT C6.

References

1. Huisgen, R. Angew. Chem. Int. Ed. (1963), 2: 565-568.
2. Rostovtsev, V.V., Green, L.G., Fokin, V.V., Sharpless, K.B. A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective Ligation of Azides and Terminal Alkynes. Angew. Chem. Int. Ed. (2002), 41: 2596-2599.
3. Green, N.M. Spectrophotometric determination of avidin and biotin. Methods Enzymol. (1970), 18A: 418-424.
4. Hirsch, J.D., Eslamizar, L., Filanoski, B.J., Malekzadeh, N., Haugland, R.P., Beechem, J.M., Haugland, R.P. Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation. Anal. Biochem. (2002), 308: 343-357.
5. van Doom, R., Slawiak, M., Szemes, M., Dullemans, A.M., Bonants, P., Kowalchuk, G.A., Schoen, C.D. Robust Definition and Identification of Multiple Oomycetes and Fungi in Environmental Samples by Using a Novel Cleavable Padlock Probe-Based Ligation Detection Assay.Appl. Environ. Microbiol. (2009), 75: 4185-4193.
- Biotin 3'

Click here for a list of other Affinity Ligand Modifications.

Biotin is an affinity label that can be incorporated at either the 5'- or 3'-end of an oligonucleotide, or at an internal position using biotin dT or Amino bases for conjugation to biotin-NHS. Biotin has a high affinity for the bacterial protein, streptavidin, which can be conjugated to a solid support (such as magnetic beads) for use as a capture and immobilization medium for a biotinylated oligo. In the biotin phosphoramidite, the biotin is attached to a long spacer arm, which acts to minimize steric hindrance between the biotin moiety and the oligo, thereby providing streptavidin easy access to the biotin. Biotinylated oligos are most commonly used as probes or primers in a variety of in vitro and in vivo applications.

Besides their importance as nucleic acid probes, biotinylated oligonucleotides are also useful for the purification of DNA binding proteins. In this context, the biotinylated oligonucleotide can be bound to a streptavidin matrix and used for either column or spin chromatography. For isolation of DNA binding proteins, the streptavidin-biotin-oligonucleotide complex is incubated with a crude cell extract containing nuclear proteins. Following appropriate washes, the proteins that bind selectively to the oligonucleotide sequence can be eluted under conditions that disrupt the protein:DNA complex. Because the binding of biotin to streptavidin is essentially irreversible and is resistant to chaotropic agents and extremes of pH and ionic strength, the elution conditions can be relatively stringent.

Dual Biotin

Dual Biotin modification specifically can be used to add multiple biotin moieties at the 5'- or 3'-end of an oligo. The most common use of this modification is to incorporate two biotin (Dual Biotin) molecules in sequence (separated by a six-carbon linker) at the 5'-end of an oligo. This "Dual Biotin" has higher binding affinity for streptavidin than that of a single biotin. The additional binding strength can be critical for applications requiring the use of biotinylated DNA attached to streptavidin-coated beads at higher temperature (for example, in PCR). Dual Biotin is known to prevent or effectively reduce loss of biotinylated DNA from such beads during heating (1). Dual biotin also is used to label the linker primers in Serial Analysis of Gene Expression (SAGE) protocols (2).

For direct biotin-labeling of target RNA transcripts for microarray analysis, a special 3'-biotinylated donor nucleotide molecule containing three biotin molecules in sequence was synthesized and then ligated to the target RNA using T4 RNA ligase. The attachment of three biotins to RNA in this manner resulted in a 30% increase in target signal intensity and improved transcript detection sensitivity (3)

.

Multi Biotin

Multi-Biotin is a modification that can be added sequentially as many units as desired based on the application. Use Dual Biotin modification code [Bio-Dual] that is specific for two units of biotin.

For direct biotin-labeling of target RNA transcripts for microarray analysis, a special 3'-biotinylated donor nucleotide molecule containing three biotin molecules in sequence was synthesized and then ligated to the target RNA using T4 RNA ligase. The attachment of three biotins to RNA in this manner resulted in a 30% increase in target signal intensity and improved transcript detection sensitivity (3)

.

Desthiobiotin

Desthiobiotin is a biotin derivative. Like biotin, desthiobiotin binds to streptavidin, but its binding affinity is considerably less (2x10E-9 M) than that of biotin (4.0x10E-14 M) (1). Consequently, oligonucleotides labeled with desthiobiotin can be easily displaced from streptavidin by biotin, thereby making recovery of the labeled oligo (for example, in affinity purification protocols) from a streptavidin-coated support a relatively simple process (2). Desthiobiotin-labeled oligos can also be conveniently eluted from streptavidin-coated supports by incubation in distilled water at 95C for 10 minutes (3). Gene Link recommends substitution of of desthiobiotin for biotin for those cases where reversible capture of oligonucleotides is desirable. Note that since desthiobiotin is in the form of an NHS ester, an active primary amino group (such as Amino Linker C6) must first be incorporated into the oligonucleotide, to allow for subsequent conjugation to desthiobiotin NHS ester.

Desthiobiotin NHS modification is a post synthesis conjugation to a primary amino group thus an additional modification with an amino group is required. A C6 or C12 amino group can be placed at the 5' or for the 3' end a C3 or C7 amino and for internal positions an amino modified base is used, e.g Amino dT C6.

References

1. Huisgen, R. Angew. Chem. Int. Ed. (1963), 2: 565-568.
2. Rostovtsev, V.V., Green, L.G., Fokin, V.V., Sharpless, K.B. A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective Ligation of Azides and Terminal Alkynes. Angew. Chem. Int. Ed. (2002), 41: 2596-2599.
3. Green, N.M. Spectrophotometric determination of avidin and biotin. Methods Enzymol. (1970), 18A: 418-424.
4. Hirsch, J.D., Eslamizar, L., Filanoski, B.J., Malekzadeh, N., Haugland, R.P., Beechem, J.M., Haugland, R.P. Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation. Anal. Biochem. (2002), 308: 343-357.
5. van Doom, R., Slawiak, M., Szemes, M., Dullemans, A.M., Bonants, P., Kowalchuk, G.A., Schoen, C.D. Robust Definition and Identification of Multiple Oomycetes and Fungi in Environmental Samples by Using a Novel Cleavable Padlock Probe-Based Ligation Detection Assay.Appl. Environ. Microbiol. (2009), 75: 4185-4193.
- Biotin deoxythymidine dT

Click here for a list of other Affinity Ligand Modifications.

Multiple Site Note: Multi Biotin structure example shown above is for three consecutive additions. Multi Biotin sites can be added sequentially as multiple units as desired. Please indicate on your order request the number of units to be added. The code [Bio-Multi] must be added in multiples to reflect the number of sites. Example for Dual Biotin the code must be entered twice; [Bio-Multi] [Bio-Multi] and for Triple Biotin it will be [Bio-Multi] [Bio-Multi] [Bio-Multi] in the sequence where desired.

Biotin is an affinity label that can be incorporated at either the 5'- or 3'-end of an oligonucleotide, or at an internal position using biotin dT or Amino bases for conjugation to biotin-NHS. Biotin has a high affinity for the bacterial protein, streptavidin, which can be conjugated to a solid support (such as magnetic beads) for use as a capture and immobilization medium for a biotinylated oligo. In the biotin phosphoramidite, the biotin is attached to a long spacer arm, which acts to minimize steric hindrance between the biotin moiety and the oligo, thereby providing streptavidin easy access to the biotin. Biotinylated oligos are most commonly used as probes or primers in a variety of in vitro and in vivo applications.

Besides their importance as nucleic acid probes, biotinylated oligonucleotides are also useful for the purification of DNA binding proteins. In this context, the biotinylated oligonucleotide can be bound to a streptavidin matrix and used for either column or spin chromatography. For isolation of DNA binding proteins, the streptavidin-biotin-oligonucleotide complex is incubated with a crude cell extract containing nuclear proteins. Following appropriate washes, the proteins that bind selectively to the oligonucleotide sequence can be eluted under conditions that disrupt the protein:DNA complex. Because the binding of biotin to streptavidin is essentially irreversible and is resistant to chaotropic agents and extremes of pH and ionic strength, the elution conditions can be relatively stringent.

Dual Biotin

Dual Biotin modification specifically can be used to add multiple biotin moieties at the 5'- or 3'-end of an oligo. The most common use of this modification is to incorporate two biotin (Dual Biotin) molecules in sequence (separated by a six-carbon linker) at the 5'-end of an oligo. This "Dual Biotin" has higher binding affinity for streptavidin than that of a single biotin. The additional binding strength can be critical for applications requiring the use of biotinylated DNA attached to streptavidin-coated beads at higher temperature (for example, in PCR). Dual Biotin is known to prevent or effectively reduce loss of biotinylated DNA from such beads during heating (1). Dual biotin also is used to label the linker primers in Serial Analysis of Gene Expression (SAGE) protocols (2).

For direct biotin-labeling of target RNA transcripts for microarray analysis, a special 3'-biotinylated donor nucleotide molecule containing three biotin molecules in sequence was synthesized and then ligated to the target RNA using T4 RNA ligase. The attachment of three biotins to RNA in this manner resulted in a 30% increase in target signal intensity and improved transcript detection sensitivity (3)

.

Multi Biotin

Multi-Biotin is a modification that can be added sequentially as many units as desired based on the application. Use Dual Biotin modification code [Bio-Dual] that is specific for two units of biotin.

For direct biotin-labeling of target RNA transcripts for microarray analysis, a special 3'-biotinylated donor nucleotide molecule containing three biotin molecules in sequence was synthesized and then ligated to the target RNA using T4 RNA ligase. The attachment of three biotins to RNA in this manner resulted in a 30% increase in target signal intensity and improved transcript detection sensitivity (3)

.

Desthiobiotin

Desthiobiotin is a biotin derivative. Like biotin, desthiobiotin binds to streptavidin, but its binding affinity is considerably less (2x10E-9 M) than that of biotin (4.0x10E-14 M) (1). Consequently, oligonucleotides labeled with desthiobiotin can be easily displaced from streptavidin by biotin, thereby making recovery of the labeled oligo (for example, in affinity purification protocols) from a streptavidin-coated support a relatively simple process (2). Desthiobiotin-labeled oligos can also be conveniently eluted from streptavidin-coated supports by incubation in distilled water at 95C for 10 minutes (3). Gene Link recommends substitution of of desthiobiotin for biotin for those cases where reversible capture of oligonucleotides is desirable. Note that since desthiobiotin is in the form of an NHS ester, an active primary amino group (such as Amino Linker C6) must first be incorporated into the oligonucleotide, to allow for subsequent conjugation to desthiobiotin NHS ester.

Desthiobiotin NHS modification is a post synthesis conjugation to a primary amino group thus an additional modification with an amino group is required. A C6 or C12 amino group can be placed at the 5' or for the 3' end a C3 or C7 amino and for internal positions an amino modified base is used, e.g Amino dT C6.

References

1. Huisgen, R. Angew. Chem. Int. Ed. (1963), 2: 565-568.
2. Rostovtsev, V.V., Green, L.G., Fokin, V.V., Sharpless, K.B. A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective Ligation of Azides and Terminal Alkynes. Angew. Chem. Int. Ed. (2002), 41: 2596-2599.
3. Green, N.M. Spectrophotometric determination of avidin and biotin. Methods Enzymol. (1970), 18A: 418-424.
4. Hirsch, J.D., Eslamizar, L., Filanoski, B.J., Malekzadeh, N., Haugland, R.P., Beechem, J.M., Haugland, R.P. Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation. Anal. Biochem. (2002), 308: 343-357.
5. van Doom, R., Slawiak, M., Szemes, M., Dullemans, A.M., Bonants, P., Kowalchuk, G.A., Schoen, C.D. Robust Definition and Identification of Multiple Oomycetes and Fungi in Environmental Samples by Using a Novel Cleavable Padlock Probe-Based Ligation Detection Assay.Appl. Environ. Microbiol. (2009), 75: 4185-4193.
- Biotin Multi

Click here for a list of other Affinity Ligand Modifications.

Biotin-NHS is an N-hydroxysuccinimide ester (NHS ester) of biotin. Biotin-NHS can be used to internally label an oligonucleotide with biotin at any base (that is, at a G, C, T or A position). To accomplish this, amino dG-C6/dC-C6/dA-C6/dT is first incorporated into the oligonucleotide, thereby placing an active primary amino group at the desired position. Biotin-NHS is then conjugated to the amino group in a separate reaction to form the final biotin-labeled product.

YIELD
NHS based modifications are post synthesis conjugation performed using a primary amino group. The yield is lower as compared to direct automated coupling of modifications that are available as amidites. Approximate yield for various scales are given below.

~2 nmol final yield for 50 nmol scale synthesis.
~5 nmol final yield for 200 nmol scale synthesis.
~16 nmol final yield for 1 umol scale synthesis.
~30 nmol final yield for 2 umol scale synthesis.
~75 nmol final yield for 5 umol scale synthesis.
~150 nmol final yield for 10 umol scale synthesis.
~225 nmol final yield for 15 umol scale synthesis.

Biotin is an affinity label that can be incorporated at either the 5'or 3'end of an oligonucleotide, or at an internal position. Biotin has a high affinity for the bacterial protein, streptavidin, which can be conjugated to a solid support (such as magnetic beads) for use as a capture and immobilization medium for a biotinylated oligo. In the biotin phosphoramidite, the biotin is attached to a long spacer arm, which acts to minimize steric hindrance between the biotin moiety and the oligo, thereby providing streptavidin easy access to the biotin. Biotinylated oligos are most commonly used as probes or primers in a variety of in vitro and in vivo applications.

Besides their importance as nucleic acid probes, biotinylated oligonucleotides are also useful for the purification of DNA binding proteins. In this context, the biotinylated oligonucleotide can be bound to a streptavidin matrix and used for either column or spin chromatography. For isolation of DNA binding proteins, the streptavidin-biotin-oligonucleotide complex is incubated with a crude cell extract containing nuclear proteins. Following appropriate washes, the proteins that bind selectively to the oligonucleotide sequence can be eluted under conditions that disrupt the protein:DNA complex. Because the binding of biotin to streptavidin is essentially irreversible and is resistant to chaotropic agents and extremes of pH and ionic strength, the elution conditions can be relatively stringent.
Biotin-NHS can also be used to biotinylate a large amount of oligonucleotide aminated at the 5'or 3'end, in aqueous solution and at relatively low cost (1).

-

The primary amine labelled oligos can also be conjugated to carboxyl functional groups usually for solid supports applications using EDC mediated reaction as shown in the figure below.

References
1. Bengtstron, M., Jungell-nortamo, A., Syvanen, A-C. Biotinylation of Oligonucleotides Using a Water Soluble Biotin Ester.Nucleos. Nucleot. Nucl. (1990), 9: 123-127.
- Biotin NHS

Click here for a list of other Affinity Ligand Modifications.

Biotin is an affinity label that can be incorporated at either the 5'- or 3'-end of an oligonucleotide, or at an internal position using biotin dT or Amino bases for conjugation to biotin-NHS. Biotin has a high affinity for the bacterial protein, streptavidin, which can be conjugated to a solid support (such as magnetic beads) for use as a capture and immobilization medium for a biotinylated oligo. In the biotin phosphoramidite, the biotin is attached to a long spacer arm, which acts to minimize steric hindrance between the biotin moiety and the oligo, thereby providing streptavidin easy access to the biotin. Biotinylated oligos are most commonly used as probes or primers in a variety of in vitro and in vivo applications.

Besides their importance as nucleic acid probes, biotinylated oligonucleotides are also useful for the purification of DNA binding proteins. In this context, the biotinylated oligonucleotide can be bound to a streptavidin matrix and used for either column or spin chromatography. For isolation of DNA binding proteins, the streptavidin-biotin-oligonucleotide complex is incubated with a crude cell extract containing nuclear proteins. Following appropriate washes, the proteins that bind selectively to the oligonucleotide sequence can be eluted under conditions that disrupt the protein:DNA complex. Because the binding of biotin to streptavidin is essentially irreversible and is resistant to chaotropic agents and extremes of pH and ionic strength, the elution conditions can be relatively stringent.

Dual Biotin

Dual Biotin modification specifically can be used to add multiple biotin moieties at the 5'- or 3'-end of an oligo. The most common use of this modification is to incorporate two biotin (Dual Biotin) molecules in sequence (separated by a six-carbon linker) at the 5'-end of an oligo. This "Dual Biotin" has higher binding affinity for streptavidin than that of a single biotin. The additional binding strength can be critical for applications requiring the use of biotinylated DNA attached to streptavidin-coated beads at higher temperature (for example, in PCR). Dual Biotin is known to prevent or effectively reduce loss of biotinylated DNA from such beads during heating (1). Dual biotin also is used to label the linker primers in Serial Analysis of Gene Expression (SAGE) protocols (2).

For direct biotin-labeling of target RNA transcripts for microarray analysis, a special 3'-biotinylated donor nucleotide molecule containing three biotin molecules in sequence was synthesized and then ligated to the target RNA using T4 RNA ligase. The attachment of three biotins to RNA in this manner resulted in a 30% increase in target signal intensity and improved transcript detection sensitivity (3)

.

Multi Biotin

Multi-Biotin is a modification that can be added sequentially as many units as desired based on the application. Use Dual Biotin modification code [Bio-Dual] that is specific for two units of biotin.

For direct biotin-labeling of target RNA transcripts for microarray analysis, a special 3'-biotinylated donor nucleotide molecule containing three biotin molecules in sequence was synthesized and then ligated to the target RNA using T4 RNA ligase. The attachment of three biotins to RNA in this manner resulted in a 30% increase in target signal intensity and improved transcript detection sensitivity (3)

.

Desthiobiotin

Desthiobiotin is a biotin derivative. Like biotin, desthiobiotin binds to streptavidin, but its binding affinity is considerably less (2x10E-9 M) than that of biotin (4.0x10E-14 M) (1). Consequently, oligonucleotides labeled with desthiobiotin can be easily displaced from streptavidin by biotin, thereby making recovery of the labeled oligo (for example, in affinity purification protocols) from a streptavidin-coated support a relatively simple process (2). Desthiobiotin-labeled oligos can also be conveniently eluted from streptavidin-coated supports by incubation in distilled water at 95C for 10 minutes (3). Gene Link recommends substitution of of desthiobiotin for biotin for those cases where reversible capture of oligonucleotides is desirable. Note that since desthiobiotin is in the form of an NHS ester, an active primary amino group (such as Amino Linker C6) must first be incorporated into the oligonucleotide, to allow for subsequent conjugation to desthiobiotin NHS ester.

Desthiobiotin NHS modification is a post synthesis conjugation to a primary amino group thus an additional modification with an amino group is required. A C6 or C12 amino group can be placed at the 5' or for the 3' end a C3 or C7 amino and for internal positions an amino modified base is used, e.g Amino dT C6.

References

1. Huisgen, R. Angew. Chem. Int. Ed. (1963), 2: 565-568.
2. Rostovtsev, V.V., Green, L.G., Fokin, V.V., Sharpless, K.B. A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective Ligation of Azides and Terminal Alkynes. Angew. Chem. Int. Ed. (2002), 41: 2596-2599.
3. Green, N.M. Spectrophotometric determination of avidin and biotin. Methods Enzymol. (1970), 18A: 418-424.
4. Hirsch, J.D., Eslamizar, L., Filanoski, B.J., Malekzadeh, N., Haugland, R.P., Beechem, J.M., Haugland, R.P. Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation. Anal. Biochem. (2002), 308: 343-357.
5. van Doom, R., Slawiak, M., Szemes, M., Dullemans, A.M., Bonants, P., Kowalchuk, G.A., Schoen, C.D. Robust Definition and Identification of Multiple Oomycetes and Fungi in Environmental Samples by Using a Novel Cleavable Padlock Probe-Based Ligation Detection Assay.Appl. Environ. Microbiol. (2009), 75: 4185-4193.
- Biotin TEG (15 atom triethylene glycol spacer) 3'

Click here for a list of other Affinity Ligand Modifications.

Biotin is an affinity label that can be incorporated at either the 5'- or 3'-end of an oligonucleotide, or at an internal position using biotin dT or Amino bases for conjugation to biotin-NHS. Biotin has a high affinity for the bacterial protein, streptavidin, which can be conjugated to a solid support (such as magnetic beads) for use as a capture and immobilization medium for a biotinylated oligo. In the biotin phosphoramidite, the biotin is attached to a long spacer arm, which acts to minimize steric hindrance between the biotin moiety and the oligo, thereby providing streptavidin easy access to the biotin. Biotinylated oligos are most commonly used as probes or primers in a variety of in vitro and in vivo applications.

Besides their importance as nucleic acid probes, biotinylated oligonucleotides are also useful for the purification of DNA binding proteins. In this context, the biotinylated oligonucleotide can be bound to a streptavidin matrix and used for either column or spin chromatography. For isolation of DNA binding proteins, the streptavidin-biotin-oligonucleotide complex is incubated with a crude cell extract containing nuclear proteins. Following appropriate washes, the proteins that bind selectively to the oligonucleotide sequence can be eluted under conditions that disrupt the protein:DNA complex. Because the binding of biotin to streptavidin is essentially irreversible and is resistant to chaotropic agents and extremes of pH and ionic strength, the elution conditions can be relatively stringent.

Dual Biotin

Dual Biotin modification specifically can be used to add multiple biotin moieties at the 5'- or 3'-end of an oligo. The most common use of this modification is to incorporate two biotin (Dual Biotin) molecules in sequence (separated by a six-carbon linker) at the 5'-end of an oligo. This "Dual Biotin" has higher binding affinity for streptavidin than that of a single biotin. The additional binding strength can be critical for applications requiring the use of biotinylated DNA attached to streptavidin-coated beads at higher temperature (for example, in PCR). Dual Biotin is known to prevent or effectively reduce loss of biotinylated DNA from such beads during heating (1). Dual biotin also is used to label the linker primers in Serial Analysis of Gene Expression (SAGE) protocols (2).

For direct biotin-labeling of target RNA transcripts for microarray analysis, a special 3'-biotinylated donor nucleotide molecule containing three biotin molecules in sequence was synthesized and then ligated to the target RNA using T4 RNA ligase. The attachment of three biotins to RNA in this manner resulted in a 30% increase in target signal intensity and improved transcript detection sensitivity (3)

.

Multi Biotin

Multi-Biotin is a modification that can be added sequentially as many units as desired based on the application. Use Dual Biotin modification code [Bio-Dual] that is specific for two units of biotin.

For direct biotin-labeling of target RNA transcripts for microarray analysis, a special 3'-biotinylated donor nucleotide molecule containing three biotin molecules in sequence was synthesized and then ligated to the target RNA using T4 RNA ligase. The attachment of three biotins to RNA in this manner resulted in a 30% increase in target signal intensity and improved transcript detection sensitivity (3)

.

Desthiobiotin

Desthiobiotin is a biotin derivative. Like biotin, desthiobiotin binds to streptavidin, but its binding affinity is considerably less (2x10E-9 M) than that of biotin (4.0x10E-14 M) (1). Consequently, oligonucleotides labeled with desthiobiotin can be easily displaced from streptavidin by biotin, thereby making recovery of the labeled oligo (for example, in affinity purification protocols) from a streptavidin-coated support a relatively simple process (2). Desthiobiotin-labeled oligos can also be conveniently eluted from streptavidin-coated supports by incubation in distilled water at 95C for 10 minutes (3). Gene Link recommends substitution of of desthiobiotin for biotin for those cases where reversible capture of oligonucleotides is desirable. Note that since desthiobiotin is in the form of an NHS ester, an active primary amino group (such as Amino Linker C6) must first be incorporated into the oligonucleotide, to allow for subsequent conjugation to desthiobiotin NHS ester.

Desthiobiotin NHS modification is a post synthesis conjugation to a primary amino group thus an additional modification with an amino group is required. A C6 or C12 amino group can be placed at the 5' or for the 3' end a C3 or C7 amino and for internal positions an amino modified base is used, e.g Amino dT C6.

References

1. Huisgen, R. Angew. Chem. Int. Ed. (1963), 2: 565-568.
2. Rostovtsev, V.V., Green, L.G., Fokin, V.V., Sharpless, K.B. A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective Ligation of Azides and Terminal Alkynes. Angew. Chem. Int. Ed. (2002), 41: 2596-2599.
3. Green, N.M. Spectrophotometric determination of avidin and biotin. Methods Enzymol. (1970), 18A: 418-424.
4. Hirsch, J.D., Eslamizar, L., Filanoski, B.J., Malekzadeh, N., Haugland, R.P., Beechem, J.M., Haugland, R.P. Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation. Anal. Biochem. (2002), 308: 343-357.
5. van Doom, R., Slawiak, M., Szemes, M., Dullemans, A.M., Bonants, P., Kowalchuk, G.A., Schoen, C.D. Robust Definition and Identification of Multiple Oomycetes and Fungi in Environmental Samples by Using a Novel Cleavable Padlock Probe-Based Ligation Detection Assay.Appl. Environ. Microbiol. (2009), 75: 4185-4193.
- Biotin TEG (15 atom triethylene glycol spacer) 5'

Click here for a list of other Affinity Ligand Modifications.

Biotin is an affinity label that can be incorporated at either the 5'- or 3'-end of an oligonucleotide, or at an internal position using biotin dT or Amino bases for conjugation to biotin-NHS. Biotin has a high affinity for the bacterial protein, streptavidin, which can be conjugated to a solid support (such as magnetic beads) for use as a capture and immobilization medium for a biotinylated oligo. In the biotin phosphoramidite, the biotin is attached to a long spacer arm, which acts to minimize steric hindrance between the biotin moiety and the oligo, thereby providing streptavidin easy access to the biotin. Biotinylated oligos are most commonly used as probes or primers in a variety of in vitro and in vivo applications.

Besides their importance as nucleic acid probes, biotinylated oligonucleotides are also useful for the purification of DNA binding proteins. In this context, the biotinylated oligonucleotide can be bound to a streptavidin matrix and used for either column or spin chromatography. For isolation of DNA binding proteins, the streptavidin-biotin-oligonucleotide complex is incubated with a crude cell extract containing nuclear proteins. Following appropriate washes, the proteins that bind selectively to the oligonucleotide sequence can be eluted under conditions that disrupt the protein:DNA complex. Because the binding of biotin to streptavidin is essentially irreversible and is resistant to chaotropic agents and extremes of pH and ionic strength, the elution conditions can be relatively stringent.

Dual Biotin

Dual Biotin modification specifically can be used to add multiple biotin moieties at the 5'- or 3'-end of an oligo. The most common use of this modification is to incorporate two biotin (Dual Biotin) molecules in sequence (separated by a six-carbon linker) at the 5'-end of an oligo. This "Dual Biotin" has higher binding affinity for streptavidin than that of a single biotin. The additional binding strength can be critical for applications requiring the use of biotinylated DNA attached to streptavidin-coated beads at higher temperature (for example, in PCR). Dual Biotin is known to prevent or effectively reduce loss of biotinylated DNA from such beads during heating (1). Dual biotin also is used to label the linker primers in Serial Analysis of Gene Expression (SAGE) protocols (2).

For direct biotin-labeling of target RNA transcripts for microarray analysis, a special 3'-biotinylated donor nucleotide molecule containing three biotin molecules in sequence was synthesized and then ligated to the target RNA using T4 RNA ligase. The attachment of three biotins to RNA in this manner resulted in a 30% increase in target signal intensity and improved transcript detection sensitivity (3)

.

Multi Biotin

Multi-Biotin is a modification that can be added sequentially as many units as desired based on the application. Use Dual Biotin modification code [Bio-Dual] that is specific for two units of biotin.

For direct biotin-labeling of target RNA transcripts for microarray analysis, a special 3'-biotinylated donor nucleotide molecule containing three biotin molecules in sequence was synthesized and then ligated to the target RNA using T4 RNA ligase. The attachment of three biotins to RNA in this manner resulted in a 30% increase in target signal intensity and improved transcript detection sensitivity (3)

.

Desthiobiotin

Desthiobiotin is a biotin derivative. Like biotin, desthiobiotin binds to streptavidin, but its binding affinity is considerably less (2x10E-9 M) than that of biotin (4.0x10E-14 M) (1). Consequently, oligonucleotides labeled with desthiobiotin can be easily displaced from streptavidin by biotin, thereby making recovery of the labeled oligo (for example, in affinity purification protocols) from a streptavidin-coated support a relatively simple process (2). Desthiobiotin-labeled oligos can also be conveniently eluted from streptavidin-coated supports by incubation in distilled water at 95C for 10 minutes (3). Gene Link recommends substitution of of desthiobiotin for biotin for those cases where reversible capture of oligonucleotides is desirable. Note that since desthiobiotin is in the form of an NHS ester, an active primary amino group (such as Amino Linker C6) must first be incorporated into the oligonucleotide, to allow for subsequent conjugation to desthiobiotin NHS ester.

Desthiobiotin NHS modification is a post synthesis conjugation to a primary amino group thus an additional modification with an amino group is required. A C6 or C12 amino group can be placed at the 5' or for the 3' end a C3 or C7 amino and for internal positions an amino modified base is used, e.g Amino dT C6.

References

1. Huisgen, R. Angew. Chem. Int. Ed. (1963), 2: 565-568.
2. Rostovtsev, V.V., Green, L.G., Fokin, V.V., Sharpless, K.B. A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective Ligation of Azides and Terminal Alkynes. Angew. Chem. Int. Ed. (2002), 41: 2596-2599.
3. Green, N.M. Spectrophotometric determination of avidin and biotin. Methods Enzymol. (1970), 18A: 418-424.
4. Hirsch, J.D., Eslamizar, L., Filanoski, B.J., Malekzadeh, N., Haugland, R.P., Beechem, J.M., Haugland, R.P. Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation. Anal. Biochem. (2002), 308: 343-357.
5. van Doom, R., Slawiak, M., Szemes, M., Dullemans, A.M., Bonants, P., Kowalchuk, G.A., Schoen, C.D. Robust Definition and Identification of Multiple Oomycetes and Fungi in Environmental Samples by Using a Novel Cleavable Padlock Probe-Based Ligation Detection Assay.Appl. Environ. Microbiol. (2009), 75: 4185-4193.
- Biotin TEG (15 atom triethylene glycol spacer) Internal

Click here for a list of other Affinity Ligand Modifications.

Biotin is an affinity label that can be incorporated at either the 5'- or 3'-end of an oligonucleotide, or at an internal position using biotin dT or Amino bases for conjugation to biotin-NHS. Biotin has a high affinity for the bacterial protein, streptavidin, which can be conjugated to a solid support (such as magnetic beads) for use as a capture and immobilization medium for a biotinylated oligo. In the biotin phosphoramidite, the biotin is attached to a long spacer arm, which acts to minimize steric hindrance between the biotin moiety and the oligo, thereby providing streptavidin easy access to the biotin. Biotinylated oligos are most commonly used as probes or primers in a variety of in vitro and in vivo applications.

Besides their importance as nucleic acid probes, biotinylated oligonucleotides are also useful for the purification of DNA binding proteins. In this context, the biotinylated oligonucleotide can be bound to a streptavidin matrix and used for either column or spin chromatography. For isolation of DNA binding proteins, the streptavidin-biotin-oligonucleotide complex is incubated with a crude cell extract containing nuclear proteins. Following appropriate washes, the proteins that bind selectively to the oligonucleotide sequence can be eluted under conditions that disrupt the protein:DNA complex. Because the binding of biotin to streptavidin is essentially irreversible and is resistant to chaotropic agents and extremes of pH and ionic strength, the elution conditions can be relatively stringent.

Dual Biotin

Dual Biotin modification specifically can be used to add multiple biotin moieties at the 5'- or 3'-end of an oligo. The most common use of this modification is to incorporate two biotin (Dual Biotin) molecules in sequence (separated by a six-carbon linker) at the 5'-end of an oligo. This "Dual Biotin" has higher binding affinity for streptavidin than that of a single biotin. The additional binding strength can be critical for applications requiring the use of biotinylated DNA attached to streptavidin-coated beads at higher temperature (for example, in PCR). Dual Biotin is known to prevent or effectively reduce loss of biotinylated DNA from such beads during heating (1). Dual biotin also is used to label the linker primers in Serial Analysis of Gene Expression (SAGE) protocols (2).

For direct biotin-labeling of target RNA transcripts for microarray analysis, a special 3'-biotinylated donor nucleotide molecule containing three biotin molecules in sequence was synthesized and then ligated to the target RNA using T4 RNA ligase. The attachment of three biotins to RNA in this manner resulted in a 30% increase in target signal intensity and improved transcript detection sensitivity (3)

.

Multi Biotin

Multi-Biotin is a modification that can be added sequentially as many units as desired based on the application. Use Dual Biotin modification code [Bio-Dual] that is specific for two units of biotin.

For direct biotin-labeling of target RNA transcripts for microarray analysis, a special 3'-biotinylated donor nucleotide molecule containing three biotin molecules in sequence was synthesized and then ligated to the target RNA using T4 RNA ligase. The attachment of three biotins to RNA in this manner resulted in a 30% increase in target signal intensity and improved transcript detection sensitivity (3)

.

Desthiobiotin

Desthiobiotin is a biotin derivative. Like biotin, desthiobiotin binds to streptavidin, but its binding affinity is considerably less (2x10E-9 M) than that of biotin (4.0x10E-14 M) (1). Consequently, oligonucleotides labeled with desthiobiotin can be easily displaced from streptavidin by biotin, thereby making recovery of the labeled oligo (for example, in affinity purification protocols) from a streptavidin-coated support a relatively simple process (2). Desthiobiotin-labeled oligos can also be conveniently eluted from streptavidin-coated supports by incubation in distilled water at 95C for 10 minutes (3). Gene Link recommends substitution of of desthiobiotin for biotin for those cases where reversible capture of oligonucleotides is desirable. Note that since desthiobiotin is in the form of an NHS ester, an active primary amino group (such as Amino Linker C6) must first be incorporated into the oligonucleotide, to allow for subsequent conjugation to desthiobiotin NHS ester.

Desthiobiotin NHS modification is a post synthesis conjugation to a primary amino group thus an additional modification with an amino group is required. A C6 or C12 amino group can be placed at the 5' or for the 3' end a C3 or C7 amino and for internal positions an amino modified base is used, e.g Amino dT C6.

References

1. Huisgen, R. Angew. Chem. Int. Ed. (1963), 2: 565-568.
2. Rostovtsev, V.V., Green, L.G., Fokin, V.V., Sharpless, K.B. A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective Ligation of Azides and Terminal Alkynes. Angew. Chem. Int. Ed. (2002), 41: 2596-2599.
3. Green, N.M. Spectrophotometric determination of avidin and biotin. Methods Enzymol. (1970), 18A: 418-424.
4. Hirsch, J.D., Eslamizar, L., Filanoski, B.J., Malekzadeh, N., Haugland, R.P., Beechem, J.M., Haugland, R.P. Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation. Anal. Biochem. (2002), 308: 343-357.
5. van Doom, R., Slawiak, M., Szemes, M., Dullemans, A.M., Bonants, P., Kowalchuk, G.A., Schoen, C.D. Robust Definition and Identification of Multiple Oomycetes and Fungi in Environmental Samples by Using a Novel Cleavable Padlock Probe-Based Ligation Detection Assay.Appl. Environ. Microbiol. (2009), 75: 4185-4193.
- Biotin-5'

Click here for a list of other Affinity Ligand Modifications.

Desthiobiotin modification is a post synthesis conjugation to a primary amino group thus an additional modification with an amino group is required. A C6 or C12 amino group can be placed at the 5' or for the 3' end a C3 or C7 amino and for internal positions an amino modified base is used, e.g Amino dT C6.

YIELD
NHS based modifications are post synthesis conjugation performed using a primary amino group. The yield is lower as compared to direct automated coupling of modifications that are available as amidites. Approximate yield for various scales are given below.

~2 nmol final yield for 50 nmol scale synthesis.
~5 nmol final yield for 200 nmol scale synthesis.
~16 nmol final yield for 1 umol scale synthesis
~160 nmol final yield for 10 umol scale synthesis
~240 nmol final yield for 15 umol scale synthesis

Desthiobiotin NHS & Desthiobiotin TEG

Desthiobiotin is a biotin derivative. Like biotin, desthiobiotin binds to streptavidin, but its binding affinity is considerably less (2x10E-9 M) than that of biotin (4.0x10E-14 M) (1). Consequently, oligonucleotides labeled with desthiobiotin can be easily displaced from streptavidin by biotin, thereby making recovery of the labeled oligo (for example, in affinity purification protocols) from a streptavidin-coated support a relatively simple process (2). Desthiobiotin-labeled oligos can also be conveniently eluted from streptavidin-coated supports by incubation in distilled water at 95C for 10 minutes (3). Gene Link recommends substitution of of desthiobiotin for biotin for those cases where reversible capture of oligonucleotides is desirable.
Desthiobiotin TEG is incorporated directly in an oligo and is available for 5' and 3'. Incorporation is also possible internally but it will break the nucleic acid base chain.

. Note that since Desthiobiotin NHS is in the form of an NHS ester, an active primary amino group (such as Amino Linker C6) must first be incorporated into the oligonucleotide, to allow for subsequent conjugation to desthiobiotin NHS ester..

Biotin

Biotin is an affinity label that can be incorporated at either the 5'- or 3'-end of an oligonucleotide, or at an internal position using biotin dT or Amino bases for conjugation to biotin-NHS. Biotin has a high affinity for the bacterial protein, streptavidin, which can be conjugated to a solid support (such as magnetic beads) for use as a capture and immobilization medium for a biotinylated oligo. In the biotin phosphoramidite, the biotin is attached to a long spacer arm, which acts to minimize steric hindrance between the biotin moiety and the oligo, thereby providing streptavidin easy access to the biotin. Biotinylated oligos are most commonly used as probes or primers in a variety of in vitro and in vivo applications.

Besides their importance as nucleic acid probes, biotinylated oligonucleotides are also useful for the purification of DNA binding proteins. In this context, the biotinylated oligonucleotide can be bound to a streptavidin matrix and used for either column or spin chromatography. For isolation of DNA binding proteins, the streptavidin-biotin-oligonucleotide complex is incubated with a crude cell extract containing nuclear proteins. Following appropriate washes, the proteins that bind selectively to the oligonucleotide sequence can be eluted under conditions that disrupt the protein:DNA complex. Because the binding of biotin to streptavidin is essentially irreversible and is resistant to chaotropic agents and extremes of pH and ionic strength, the elution conditions can be relatively stringent.

Dual Biotin

Dual Biotin modification specifically can be used to add multiple biotin moieties at the 5'- or 3'-end of an oligo. The most common use of this modification is to incorporate two biotin (Dual Biotin) molecules in sequence (separated by a six-carbon linker) at the 5'-end of an oligo. This "Dual Biotin" has higher binding affinity for streptavidin than that of a single biotin. The additional binding strength can be critical for applications requiring the use of biotinylated DNA attached to streptavidin-coated beads at higher temperature (for example, in PCR). Dual Biotin is known to prevent or effectively reduce loss of biotinylated DNA from such beads during heating (1). Dual biotin also is used to label the linker primers in Serial Analysis of Gene Expression (SAGE) protocols (2).

For direct biotin-labeling of target RNA transcripts for microarray analysis, a special 3'-biotinylated donor nucleotide molecule containing three biotin molecules in sequence was synthesized and then ligated to the target RNA using T4 RNA ligase. The attachment of three biotins to RNA in this manner resulted in a 30% increase in target signal intensity and improved transcript detection sensitivity (3)

.

Multi Biotin

Multi-Biotin is a modification that can be added sequentially as many units as desired based on the application. Use Dual Biotin modification code [Bio-Dual] that is specific for two units of biotin.

For direct biotin-labeling of target RNA transcripts for microarray analysis, a special 3'-biotinylated donor nucleotide molecule containing three biotin molecules in sequence was synthesized and then ligated to the target RNA using T4 RNA ligase. The attachment of three biotins to RNA in this manner resulted in a 30% increase in target signal intensity and improved transcript detection sensitivity (3)

.

References

1. Huisgen, R. Angew. Chem. Int. Ed. (1963), 2: 565-568.
2. Rostovtsev, V.V., Green, L.G., Fokin, V.V., Sharpless, K.B. A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective Ligation of Azides and Terminal Alkynes. Angew. Chem. Int. Ed. (2002), 41: 2596-2599.
3. Green, N.M. Spectrophotometric determination of avidin and biotin. Methods Enzymol. (1970), 18A: 418-424.
4. Hirsch, J.D., Eslamizar, L., Filanoski, B.J., Malekzadeh, N., Haugland, R.P., Beechem, J.M., Haugland, R.P. Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation. Anal. Biochem. (2002), 308: 343-357.
5. van Doom, R., Slawiak, M., Szemes, M., Dullemans, A.M., Bonants, P., Kowalchuk, G.A., Schoen, C.D. Robust Definition and Identification of Multiple Oomycetes and Fungi in Environmental Samples by Using a Novel Cleavable Padlock Probe-Based Ligation Detection Assay.Appl. Environ. Microbiol. (2009), 75: 4185-4193.
- Desthiobiotin NHS

Click here for a list of other Affinity Ligand Modifications.

Desthiobiotin

Desthiobiotin is a biotin derivative. Like biotin, desthiobiotin binds to streptavidin, but its binding affinity is considerably less (2x10E-9 M) than that of biotin (4.0x10E-14 M) (1). Consequently, oligonucleotides labeled with desthiobiotin can be easily displaced from streptavidin by biotin, thereby making recovery of the labeled oligo (for example, in affinity purification protocols) from a streptavidin-coated support a relatively simple process (2). Desthiobiotin-labeled oligos can also be conveniently eluted from streptavidin-coated supports by incubation in distilled water at 95C for 10 minutes (3). Gene Link recommends substitution of of desthiobiotin for biotin for those cases where reversible capture of oligonucleotides is desirable. Note that since desthiobiotin is in the form of an NHS ester, an active primary amino group (such as Amino Linker C6) must first be incorporated into the oligonucleotide, to allow for subsequent conjugation to desthiobiotin NHS ester.

Desthiobiotin TEG is incorporated directly in an oligo and is available for 5' and 3'. Incorporation is also possible internally but it will break the nucleic acid base chain.

Biotin

Biotin is an affinity label that can be incorporated at either the 5'- or 3'-end of an oligonucleotide, or at an internal position using biotin dT or Amino bases for conjugation to biotin-NHS. Biotin has a high affinity for the bacterial protein, streptavidin, which can be conjugated to a solid support (such as magnetic beads) for use as a capture and immobilization medium for a biotinylated oligo. In the biotin phosphoramidite, the biotin is attached to a long spacer arm, which acts to minimize steric hindrance between the biotin moiety and the oligo, thereby providing streptavidin easy access to the biotin. Biotinylated oligos are most commonly used as probes or primers in a variety of in vitro and in vivo applications.

Besides their importance as nucleic acid probes, biotinylated oligonucleotides are also useful for the purification of DNA binding proteins. In this context, the biotinylated oligonucleotide can be bound to a streptavidin matrix and used for either column or spin chromatography. For isolation of DNA binding proteins, the streptavidin-biotin-oligonucleotide complex is incubated with a crude cell extract containing nuclear proteins. Following appropriate washes, the proteins that bind selectively to the oligonucleotide sequence can be eluted under conditions that disrupt the protein:DNA complex. Because the binding of biotin to streptavidin is essentially irreversible and is resistant to chaotropic agents and extremes of pH and ionic strength, the elution conditions can be relatively stringent.

Dual Biotin

Dual Biotin modification specifically can be used to add multiple biotin moieties at the 5'- or 3'-end of an oligo. The most common use of this modification is to incorporate two biotin (Dual Biotin) molecules in sequence (separated by a six-carbon linker) at the 5'-end of an oligo. This "Dual Biotin" has higher binding affinity for streptavidin than that of a single biotin. The additional binding strength can be critical for applications requiring the use of biotinylated DNA attached to streptavidin-coated beads at higher temperature (for example, in PCR). Dual Biotin is known to prevent or effectively reduce loss of biotinylated DNA from such beads during heating (1). Dual biotin also is used to label the linker primers in Serial Analysis of Gene Expression (SAGE) protocols (2).

For direct biotin-labeling of target RNA transcripts for microarray analysis, a special 3'-biotinylated donor nucleotide molecule containing three biotin molecules in sequence was synthesized and then ligated to the target RNA using T4 RNA ligase. The attachment of three biotins to RNA in this manner resulted in a 30% increase in target signal intensity and improved transcript detection sensitivity (3)

.

Multi Biotin

Multi-Biotin is a modification that can be added sequentially as many units as desired based on the application. Use Dual Biotin modification code [Bio-Dual] that is specific for two units of biotin.

For direct biotin-labeling of target RNA transcripts for microarray analysis, a special 3'-biotinylated donor nucleotide molecule containing three biotin molecules in sequence was synthesized and then ligated to the target RNA using T4 RNA ligase. The attachment of three biotins to RNA in this manner resulted in a 30% increase in target signal intensity and improved transcript detection sensitivity (3)

.

References

1. Huisgen, R. Angew. Chem. Int. Ed. (1963), 2: 565-568.
2. Rostovtsev, V.V., Green, L.G., Fokin, V.V., Sharpless, K.B. A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective Ligation of Azides and Terminal Alkynes. Angew. Chem. Int. Ed. (2002), 41: 2596-2599.
3. Green, N.M. Spectrophotometric determination of avidin and biotin. Methods Enzymol. (1970), 18A: 418-424.
4. Hirsch, J.D., Eslamizar, L., Filanoski, B.J., Malekzadeh, N., Haugland, R.P., Beechem, J.M., Haugland, R.P. Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation. Anal. Biochem. (2002), 308: 343-357.
5. van Doom, R., Slawiak, M., Szemes, M., Dullemans, A.M., Bonants, P., Kowalchuk, G.A., Schoen, C.D. Robust Definition and Identification of Multiple Oomycetes and Fungi in Environmental Samples by Using a Novel Cleavable Padlock Probe-Based Ligation Detection Assay.Appl. Environ. Microbiol. (2009), 75: 4185-4193.
- Desthiobiotin TEG

This modification is a post synthesis conjugation to a primary amino group thus an additional modification with an amino group is required. A C3, C6 or C12 amino group can be placed at the 5' or for the 3' end a C3 or C7 amino and for internal positions an amino modified base is used, e.g Amino dT C6.

Yield of Post Synthesis NHS, Maleimide & Click Ligand Conjugation*

Oligo Scale of Synthesis	Yield, nmols
50 nmol	2 nmol
200 nmol	5 nmol
1 umol	16 nmol
2 umol	30 nmol
5 umol	75 nmol
10 umol	150 nmol
15 umol	225 nmol
* The yield will be lower for oligos longer than 50mer. Click here for yield table of long oligos.
* Click here for RNA Oligos scale of synthesis and yield.
NHS Ligand conjugation requires a primary amino group. Gene Link offers a wide selection of amino modifications for 5', 3' and internal sites. Click here for a list of conjugation chemistry modifications.
Maleimide Ligand conjugation requires a thiol group. Gene Link offers a wide selection of thiol modifications for 5', 3' and internal sites. Click here for a list of conjugation chemistry modifications.
Click Chemistry Ligand conjugation requires a corresponding Click modification; examples Alkyne:Azide, Azide:DBCO, BCN:Azide, BCN:Tetrazine and TCO:Tetrazine. Gene Link offers a wide selection of click modifications for 5', 3' and internal sites. Click here for a list of click chemistry modifications.

Digoxigenin (as Digoxigenin-3-O-methylcarbonyl-epsilon-aminocaproic acid NHS ester) is a member of the steroid family found in Digitalis plants (1). It is a hapten, that is, a small molecule having high immunogenicity. Because antibodies raised against haptens have considerably higher affinities for them than other antibodies do for their targets makes haptens particularly desirable as affinity tags for oligonucleotides (2).

Digoxigenin ('Dig') is commonly used to label oligonucleotides probes for use in hybridization applications, for example, in situ hybridization (3), Northern and Southern blotting. After hybridization to their targets, these Dig-labeled probes are detected with anti-Dig antibodies that are labeled with dyes (for primary detection) or enzymes (for secondary detection using a fluorogenic, chemiluminogenic, or colorimetric substrate specific for the enzyme). To maximize signal, Gene Link recommends modifying the oligonucleotide probe with three or more Dig molecules, spaced about 10 bases apart. Note that since digoxigenin is in the form of an NHS ester, an active primary amino group (such as Amino Linker C6) must first be incorporated into the oligonucleotide, to allow for subsequent conjugation to the digoxigenin NHS ester.

References
1. Polya, G. Biochemical targets of plant bioactive compounds. New York: CRC Press, 2003. p 847.
2. Shreder, K. Synthetic Haptens as Probes of Antibody Response and Immunorecognition. Methods (Academic Press) (2000), 20: 372-379.
3. Hauptmann, G., Gerster, T.. Two-color whole-mount in situ hybridixation to vertebrate and Drosophila embryos.Trends Genet. (1994), 10: 266.
- Digoxigenin NHS

DNP (2,4-dinitrophenyl) is classified as a hapten for molecular biology purposes, that is, a small molecule having high immunogenicity. Because antibodies raised against haptens have considerably higher affinities for them than other antibodies do for their targets makes haptens particularly desirable as affinity tags for oligonucleotides (1).

DNP attached to a triethylene glycol (TEG) spacer arm is commonly used to label oligonucleotides probes for use in hybridization applications, for example, in situ hybridization, Northern and Southern blotting (2). After hybridization to their targets, these DNP-labeled probes are detected with anti-DNP antibodies that are labeled with dyes (for primary detection) or enzymes (for secondary detection using a fluorogenic, chemiluminogenic, or colorimetric (3) substrate specific for the enzyme). To maximize signal obtained with such probes, Gene Link recommends modifying the oligonucleotide probe with three DNP molecules, either grouped at the 5’-end or spaced about 10 bases apart (2).

In addition to the above straightforward anti-DNP antibody-based detection systems, oligo probes labeled with both a fluorescent dye and DNP also been used for highly-sensitive direct detection of antigens (at femtoMolar levels) in a rolling circle amplification (RCA)-based assay system (4).

References
1. Shreder, K. Synthetic Haptens as Probes of Antibody Response and Immunorecognition. Methods (Academic Press) (2000), 20: 372-379.
2. Grzybowski, J., Will, D.W., Randall, R.E., Smith, C.A., Brown, T.. Synthesis and antibody-mediated detection of oligonucleotides containing multiple 2,4-dinitrophenyl reporter groups. Nucleic Acids Res. (1993), 21: 1705-1712.
3. Lehtovaara, P., Uusi-Oukari, M., Buchert, P, Laaksonen, M., Bengtstrom, M. Ranki, M. Quantitative PCR for Hepatitis B Virus with Colorimetric Detection.Genome Res. (1993), 3: 169-175.
4. Schweitzer, B., Wiltshire, S., Lambert, J., O’Malley, S., et al. Immunoassays with rolling circle DNA amplification: A versatile platform for ultrasensitive antigen detection.Proc. Natl. Acad. Sci. (USA) (2000), 97: 10113-10119.
- DNP TEG (2, 4-dinitrophenyl)

Click here for a list of other Affinity Ligand Modifications.

Biotin is an affinity label that can be incorporated at either the 5'- or 3'-end of an oligonucleotide, or at an internal position using biotin dT or Amino bases for conjugation to biotin-NHS. Biotin has a high affinity for the bacterial protein, streptavidin, which can be conjugated to a solid support (such as magnetic beads) for use as a capture and immobilization medium for a biotinylated oligo. In the biotin phosphoramidite, the biotin is attached to a long spacer arm, which acts to minimize steric hindrance between the biotin moiety and the oligo, thereby providing streptavidin easy access to the biotin. Biotinylated oligos are most commonly used as probes or primers in a variety of in vitro and in vivo applications.

Besides their importance as nucleic acid probes, biotinylated oligonucleotides are also useful for the purification of DNA binding proteins. In this context, the biotinylated oligonucleotide can be bound to a streptavidin matrix and used for either column or spin chromatography. For isolation of DNA binding proteins, the streptavidin-biotin-oligonucleotide complex is incubated with a crude cell extract containing nuclear proteins. Following appropriate washes, the proteins that bind selectively to the oligonucleotide sequence can be eluted under conditions that disrupt the protein:DNA complex. Because the binding of biotin to streptavidin is essentially irreversible and is resistant to chaotropic agents and extremes of pH and ionic strength, the elution conditions can be relatively stringent.

Dual Biotin

Dual Biotin modification specifically can be used to add multiple biotin moieties at the 5'- or 3'-end of an oligo. The most common use of this modification is to incorporate two biotin (Dual Biotin) molecules in sequence (separated by a six-carbon linker) at the 5'-end of an oligo. This "Dual Biotin" has higher binding affinity for streptavidin than that of a single biotin. The additional binding strength can be critical for applications requiring the use of biotinylated DNA attached to streptavidin-coated beads at higher temperature (for example, in PCR). Dual Biotin is known to prevent or effectively reduce loss of biotinylated DNA from such beads during heating (1). Dual biotin also is used to label the linker primers in Serial Analysis of Gene Expression (SAGE) protocols (2).

For direct biotin-labeling of target RNA transcripts for microarray analysis, a special 3'-biotinylated donor nucleotide molecule containing three biotin molecules in sequence was synthesized and then ligated to the target RNA using T4 RNA ligase. The attachment of three biotins to RNA in this manner resulted in a 30% increase in target signal intensity and improved transcript detection sensitivity (3)

.

Multi Biotin

Multi-Biotin is a modification that can be added sequentially as many units as desired based on the application. Use Dual Biotin modification code [Bio-Dual] that is specific for two units of biotin.

For direct biotin-labeling of target RNA transcripts for microarray analysis, a special 3'-biotinylated donor nucleotide molecule containing three biotin molecules in sequence was synthesized and then ligated to the target RNA using T4 RNA ligase. The attachment of three biotins to RNA in this manner resulted in a 30% increase in target signal intensity and improved transcript detection sensitivity (3)

.

Desthiobiotin

Desthiobiotin is a biotin derivative. Like biotin, desthiobiotin binds to streptavidin, but its binding affinity is considerably less (2x10E-9 M) than that of biotin (4.0x10E-14 M) (1). Consequently, oligonucleotides labeled with desthiobiotin can be easily displaced from streptavidin by biotin, thereby making recovery of the labeled oligo (for example, in affinity purification protocols) from a streptavidin-coated support a relatively simple process (2). Desthiobiotin-labeled oligos can also be conveniently eluted from streptavidin-coated supports by incubation in distilled water at 95C for 10 minutes (3). Gene Link recommends substitution of of desthiobiotin for biotin for those cases where reversible capture of oligonucleotides is desirable. Note that since desthiobiotin is in the form of an NHS ester, an active primary amino group (such as Amino Linker C6) must first be incorporated into the oligonucleotide, to allow for subsequent conjugation to desthiobiotin NHS ester.

Desthiobiotin NHS modification is a post synthesis conjugation to a primary amino group thus an additional modification with an amino group is required. A C6 or C12 amino group can be placed at the 5' or for the 3' end a C3 or C7 amino and for internal positions an amino modified base is used, e.g Amino dT C6.

References

1. Huisgen, R. Angew. Chem. Int. Ed. (1963), 2: 565-568.
2. Rostovtsev, V.V., Green, L.G., Fokin, V.V., Sharpless, K.B. A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective Ligation of Azides and Terminal Alkynes. Angew. Chem. Int. Ed. (2002), 41: 2596-2599.
3. Green, N.M. Spectrophotometric determination of avidin and biotin. Methods Enzymol. (1970), 18A: 418-424.
4. Hirsch, J.D., Eslamizar, L., Filanoski, B.J., Malekzadeh, N., Haugland, R.P., Beechem, J.M., Haugland, R.P. Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: uses for protein labeling, detection, and isolation. Anal. Biochem. (2002), 308: 343-357.
5. van Doom, R., Slawiak, M., Szemes, M., Dullemans, A.M., Bonants, P., Kowalchuk, G.A., Schoen, C.D. Robust Definition and Identification of Multiple Oomycetes and Fungi in Environmental Samples by Using a Novel Cleavable Padlock Probe-Based Ligation Detection Assay.Appl. Environ. Microbiol. (2009), 75: 4185-4193.
- Dual Biotin

YIELD
NTA modified oligos.

~ 2 nmol final yield for 50 nmol scale synthesis.
~ 6 nmol final yield for 200 nmol scale synthesis.
~ 20 nmol final yield for 1 umol scale synthesis.

NTA-Oligo modified with nitrilotriacetate (NTA)is a post synthesis conjugation to thiol. The thiol group can be placed at the 5' and 3' and for internal positions DTSPA can be used. The price listed is for NTA modification and does not include thiol modification, oligo synthesis and purification charges

NTA-Oligo modified with nitrilotriacetate (NTA), which has high affinity to a His-tag on recombinant protein via the complexation of Ni2+.
It is a novel method to prepare a DNA-protein conjugate using histidine-tag (His-tag) chemistry.

References

1. J. Shimada, T. Maruyama, T. Hosogi, J. Tominaga, N. Kamiya, M. Goto,. Conjugation of DNA with protein using His-tag chemistry and its application to the aptamer-based detection system, Biotechnol. Lett. 30 (2008) 2001–2006.
2.J. Shimada et al DNA–enzyme conjugate that can detect thrombin. Anal. Biochem. 414 (2011) 103–108.
- NTA Mono (Nitrilotriacetate) Oligo Conjugate

Pseudouridine-("psi") is a C-glyoside isomer of uridine, and is the most common modified nucleoside found in structural RNA, such as tRNA, rRNA, snRNA, and snoRNA (1,2). Psi-modified RNA can be used as research tools for studies into the roles of this residue in RNA structure and function in the cell. Currently, the role of psi in RNA is a subject of active research, with some things now known. Psi can coordinate a water molecule through its free N1 hydrogen, thereby inducing a modest increase in rigidity on the nearby sugar-phosphate backbone. The presence of psi also enhances base-stacking. Such effects have been proposed as explanations for the deleterious functional effects observed in mutant bacterial strains that lack certain psi residues in tRNA or rRNA (2). Also, based on recent studies, it has been proposed that psi may offer RNA molecules protection from radiation (3).

References
1. Hamma, T., Ferre-D-Amare, A.R. Pseudouridine synthases. Chem. Biol. (2006), 13: 1125-1135.
2. Charette, M., Gray, M.W. Pseudouridine in RNA: what, where, how, and wny.IUBMB Life (2000), 49: 341-351.
3. Monobe, M., Arimoto-Kobayashi, S., Ando, K. beta-Pseudouridine, a beer component, reduces radiation-induced chromosome aberrations in human lymphocytes. Mut. Res-Genet. Toxicol. and Env. Mutagen. (2003), 538: 93-99.
- pseudoUridine ribo (psi rU)

Oligonucleotides are predominantly hydrophilic species and require help in permeating cell membranes. One strategy to improve cellular uptake of therapeutic oligonucleotides is to conjugate them with non-toxic, lipophilic molecules. Gene Link offers cholesteryl TEG, alpha-tocopherol and stearyl labelling of oligonucleotides and this strategy has proved to be useful for delivering therapeutic oligonucleotides to a broad distribution of targets.

Stearyl Modification
Stearyl Modification is C18 lipid, it is an economical and effective carrier molecule. We envisage that the 5'-stearyl group will become a favored lipophilic carrier for experimentation with synthetic oligonucleotides.


Cholesterol TEG Modification
Cholesterol TEG Modification is a lipophilic modification aiding in cellular delivery. The TEG liker arm facilitates solubility issues of the oligo making it soluble in aqueous buffers.


alpha-tocopherol TEG Modification
Similar to cholesterol TEG, alpha-tocopherol (vitamin E) is both lipophilic and non-toxic even at high doses so would be an excellent candidate as a lipophilic carrier for oligonucleotides. The TEG liker arm facilitates solubility issues of the oligo making it soluble in aqueous buffers.

GalNAc
A more directed approach to the delivery of therapeutic oligonucleotides specifically to the liver has been to target the asialoglycoprotein receptor (ASGPR) using a suitable glycoconjugate. Indeed, ASGPR is the ideal target for delivery of therapeutic oligonucleotides to the liver since it combines tissue specificity, high expression levels and rapid internalization and turnover. The use of oligonucleotide glycoconjugates has led to significant advances in therapeutic delivery as evidenced by the work of Alnylam Pharmaceuticals which has developed multivalent N-acetylgalactosamine (GalNAc) conjugated siRNAs that bind at nanomolar levels to ASGPR (1). A similar strategy has been applied at Ionis Pharmaceuticals directed at the development of antisense oligonucleotide therapeutics (2).
The GalNAc ligand originally used by Alnylam is the triantennary ligand would seem to lend itself to formation by post synthesis conjugation to the 3' terminus but a complex trivalent GalNAc support would also be perfectly applicable, if challenging to produce. However, an alternative approach using a monovalent GalNAc support with two additions of a monovalent GalNAc phosphoramidite was also described and yielded a trivalent GalNAc structure. This (1+1+1) trivalent GalNAc structure led to GalNAc modified siRNA oligos with potency equal to the equivalent siRNA with the triantennary GalNAc ligand both in vitro and in vivo.
Researchers at Ionis have developed antisense oligonucleotides containing the GalNAc cluster. In their case, they were able to show2 that moving the triantennary GalNAc ligand to the 5' terminus led to improved potency in vitro and in vivo. As may be expected, such a large complex ligand lends itself to solution phase chemistry to produce GalNAc modified antisense oligos. However, a solid phase synthetic approach was also described, and compared to the solution phase approach structure of the 5'-GalNAc triantennary ligand (4).
A further report on antisense oligonucleotides demonstrated (5) the effectiveness of modifying at the 5' terminus using monovalent GalNAc ligands. Up to five GalNAc monomers were added in a serial manner (Figure 3) and it was shown that activity of the antisense oligonucleotides improved as the number of GalNAc units increased. The authors also showed that phosphodiester linkages between the GalNAc units were preferable to phosphorothioate linkages in their testing (5).

Recommended Further Reading
N-acetylgalactosamine (GalNAc) Oligo Application Note: Glen Report 29.14: N-acetylgalactosamine (GalNAc) Oligonucleotide Conjugates

References. Adapted from Glen Research Reports. http://www.glenresearch.com/GlenReports/GR29-14.html
1. J.K. Nair, et al., J Am Chem Soc, 2014, 136, 16958-61.
2. T.P. Prakash, et al., Bioorganic & Medicinal Chemistry Letters, 2015, 25, 4127-4130.
3. K.G. Rajeev, et al., Chembiochem, 2015, 16, 903-8.
4. I. Cedillo, et al., Molecules, 2017, 22.
5. T. Yamamoto, M. Sawamura, F. Wada, M. Harada-Shiba, and S. Obika, Bioorganic & Medicinal Chemistry, 2016, 24, 26-32.

- Stearyl C18 LMO LMO 3'

Gene Link provides custom synthesis of NTA Tris (Nitrilotriacetate) Oligo Conjugate.
NTA-Tris modification is a post synthesis conjugation to an active NHS group of either a NHS-Carboxy C10 or a NHS-dT group thus an additional modification is required at the 5' end with additional charges for that modification.

YIELD
NHS based modifications are post synthesis conjugation performed, the yield is lower as compared to direct automated coupling of modifications that are available as amidites. Approximate yield for various scales are given below.

Yield given below are for oligos shorter than 50mer. Please see longer oligos yield at this link Long Oligo Typical Yield.

~2 nmol final yield for 50 nmol scale synthesis.
~5 nmol final yield for 200 nmol scale synthesis.
~16 nmol final yield for 1 umol scale synthesis
~32 nmol final yield for 2 umol scale synthesis
~160 nmol final yield for 10 umol scale synthesis
~240 nmol final yield for 15 umol scale synthesis

Mono-NTA has an affinity of ~10 uM towards His-tag
Tris-NTA (3 Ni-NTA) groups bind His-tag with an affinity of ~10 uM towards His-tag. This is 10.000-fold higher affinity as compared to mono-NTA.
Binding is reversible with imidazole or EDTA
Allows reversible labeling of proteins or cell surfaces
Immobilize proteins, lipids or cells to surfaces
Enables wide variety of functional assays - Tris NTA (Tris Nitrilotriacetate) Oligo Conjugate

Tyramide-conjugated DNA barcodes & Tyramide Signal Amplification

See a report by Paul Simonson, Itzel Valencia, Sanjay S. Patel. Indexable signal amplification for multiparametric imaging. in bioRxiv preprint.

Multiparametric imaging allows researchers to measure the expression of many biomarkers simultaneously, allowing detailed characterization of cell microenvironments. One such technique, CODEX, allows fluorescence imaging of >30 proteins in a single tissue section. In the commercial CODEX system, primary antibodies are conjugated to DNA barcodes. This modification can result in antibody dysfunction, and development of a custom antibody panel can be very costly and time consuming as trial and error of modified antibodies proceeds. To address these challenges, we developed novel tyramide-conjugated DNA barcodes that can be used with primary antibodies via peroxidase-conjugated secondary antibodies. This approach results in signal amplification and imaging without the need to conjugate primary antibodies. When combined with commercially available barcode-conjugated primary antibodies, working antibody panels can be quickly developed. This report presents methods to perform antibody staining using a commercially available automated tissue stainer and in situ hybridization imaging on a CODEX platform.

In this report, they authors present a generalizable method for CODEX imaging with unmodified primary antibodies and signal amplification that uses novel tyramide-conjugated DNA barcodes (tyramide-barcodes). This allows the benefits of tyramide signal amplification, but image the targets in an indexed way using a commercial CODEX imaging system. This approach is flexible, and demonstrate that it can be used with in situ hybridization probes. In order to make this system workable, they developed a staining approach that allowed to perform both tyramide-barcode staining and standard CODEX staining using a commercially available automated tissue stainer, made possible using a custom coverslip holder. This drastically reduces the amount of manual labor required for CODEX staining and has the potential to improve staining reproducibility.

- Tyramide Oligo