Custom Oligonucleotide 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 phosphoramidite - Biotin

Biotin-dT is a deoxythymidine nucleotide base attached to biotin through a long-chain alkyl spacer arm. Biotin-dT is typically used to label an oligonucleotide with biotin at an internal base position, though it can also be used for 5’- and 3’-end labeling as well. Internal biotin-labeling is necessary when an oligo needs to be captured or immobilized, but both ends are unavailable. A good example of this is the use of internal biotin-labeling to facilitate the immobilization of a molecular beacon on a glass slide (1). The internal labeling strategy can also be used to label a hybridization detection probe with several biotins to enhance detection of a target sequence with anti-biotin antibodies attached to colored particles (2), fluorescent dyes, or enzymes.

References
1. Fang, X., Liu, X., Schuster, S., Tan, W. Designing a novel molecular beacon for surface immobilized DNA hybridization studies. J. Am. Chem. Soc. (1999), 121: 2921-2922.
2. Dineva, M.A., Candotti, D., Fletcher-Brown, F., Allain, J-P., Lee, H. Simultaneous Visual Detection of Multiple Viral Amplicons by Dipstick Assay.J. Clin. Microbiol. (2005), 43: 4015-4021.
- Biotin deoxythymidine dT

"Biotin multi" 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 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)

. References
1. Dressman, D., Yan, H., Traverso, G., Kinzler, K.W., Vogelstein, B. Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations. Proc. Natl. Acad. Sci. USA. (2003), 100: 8817-8822.
2. Roh, T-Y., Zhao, K. High-resolution, Genome-Wide Mapping of Chromatin Modifications by GMAT. Methods Mol. Biol. (2007), 387: 95-108.
3. Cole, K., Truong, V., Barone, D., McGall, G. Direct labeling of RNA with multiple biotins allows sensitive expression profiling of acute leukemia class predictor genes.Nucleic Acids Res. (2004), 32(11): e86.
- Biotin multiple additions

Biotin-NHS is an N-hydroxysuccinimide ester (NHS ester) of biotin. Biotin-NHS can be used to internally label an oligonucleotide with biotin at a non-T position (that is, at a G, C, or A position). To accomplish this, amino dG-C6/dC-C6/dA-C6 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.

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).

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

Biotin-TEG is a biotin attached to a 15-atom mixed polarity triethylene glycol spacer, and can be incorporated at either the 5’- or 3’-end of an oligonucleotide, or at an internal position. The spacer acts to minimize steric hindrance between the biotin moiety and the oligo, thereby providing streptavidin easy access to the biotin for capture and immobilization of the oligo. Additional technical details for biotin are presented in the Biotin technical sheet.

Biotin TEG phosphoramidite - Biotin TEG (15 atom triethylene glycol spacer)

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.

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.

References
1. Green, N.M. Spectrophotometric determination of avidin and biotin. Methods Enzymol. (1970), 18A: 418-424.
2. 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.
3. 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

Digoxigenin 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.

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 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 NHA

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)

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 products for cholesteryl TEG [26-6602-XX], a-tocopheryl [26-6752-XX]and stearyl[26-6753-XX] labelling of oligonucleotides and this strategy has proved to be useful for delivering therapeutic oligonucleotides to a broad distribution of targets.
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).

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.

- N-Acetylgalactosamine C3 (GalNAc) siRNA

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.

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 (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)