Oligonucleotide Linkers or Adapters for Bioconjugation

Synthetic oligonucleotide linkers or adaptors have a wide application range in biochemical, biological, forensic, genetic, and medical research, and most of them are used together with the famous PCR reaction. For example, the annealing of two, well designed single-stranded adaptor molecules permits the creation of double-stranded adaptors with different restriction enzyme sites at each end allowing the joining of molecules that have nonhomologous protruding termini. However, the ligation of oligonucleotide linkers or adaptors to oligonucleotides requires the availability of an 5'-phosphate group at the site of ligation on either the linker or the DNA. Many applications for the use of oligonucleotide linkers or DNA linkers and adaptors have been reviewed in the recent literature.

Oligonucleotides, usually single-stranded DNA and RNA molecules of various sizes, are now routinely synthesized in commercial laboratories using automated solid-phase chemical synthesis and protected phophoramidites. Modern synthesis methods allow the manufacture of oligonucleotides with specific sequences including the placement of artificial nucleotides such as bridged-nucleic acids (BNAs) within the DNA or RNA sequence, which is very useful for the construction of specific probes and primers.

Examples of applications that use oligonucleotides include allele-specific oligonucleotide (ASO) analysis, antisense oligonucleotides (ASOs), antisense therapy, classical DNA sequencing and next-generation sequencing (NGS), DNA microarrays, fluorescent in situ hybridization (FISH), PCR primers, RT-PCR, Southern blottting, the synthesis of artificial genes, and many others. For most applications, for diagnostic purposes and as tracer molecules oligonucleotides are usually labeled or tagged with fluorophors or radioactive molecules.

Traditionally amino- and sulfhydryl-groups attached to oligonucleotides have been the most commonly used functional groups in bio-conjugation applications (1, 2).

This types of attachment chemistries have been adopted from peptide chemistries and became an important complement to the fast growing area of specialized oligonucleotide synthesis.

The complexity of modern synthetic biology has increased the demand for optimal attachment and labeling methods that do not interfere with biological processes. However, conventional methods are limited by the chemistries used, therefore it is important to have a larger arsenal of completely orthogonal and chemo-selective methods. That is the reason why during the last decade dozens of new attachment and labeling methods have been developed that increased the availability and versatility of conjugation tools.

Carboxyl modified oligonucleotides

Oligonucleotide conjugation is predominantly carried out by use of a nucleophilic group on an oligonucleotide to react with an electrophilic group on a reporter molecule or a solid support. This is predominant approach because the common oligonucleotide deprotection is performed by base treatment e.g. ammonia, primary alkylamines or their combinations which are inherently nucleophilic. However, there are many situations when researchers need to introduce an electrophilic group into oligonucleotides and use it in the attachment method towards of a nucleophilic moiety.

In the case, when oligonucleotides have been used in that type of the bio-conjugation the activated carboxylate have been generated post-synthetically (2). Free carboxyl modified oligonucleotides can be activated by EDC in situ in the organic or aqueous conditions and subsequently conjugated to aminated counterpart (Fig. 1). However, in order to generate free carboxylate attached oligonucleotide using commercially available building blocks it requires cleavage of the ester bond with alkaline base (4,5) before oligonucleotide base deprotection in concentrated ammonia, otherwise the ester will beconverted into corresponding amide.

Figure 1. Reaction scheme of the bio-conjugation between a carboxyl modified oligonucleotide and an alkylamino moiety using 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, EDAC or EDCI), a water soluble carbodiimide typically used in the 4.0 - 6.0 pH range, as the carboxyl activating agent.

Using the bio-conjugation strategy outlined in figure 1, carboxyl modified oligonucleotides can be easily immobilized on solid supports such as micro-array slides aa well as various other types of aminoalkylated beads.

Huisgen’s 1-3 Dipolar cycloaddition

The Copper (I) catalyzed Huisgen’s 1,3-dipolar cycloaddition between alkynes and azides discovered by the Sharpless group in 2002 (called “click chemistry”) (9), is a novel and very powerful method for the incorporation of molecules of interest such as reporter molecules, lipophilic ligands, and others, into oligonucleotides. The methods have been limited to the post-synthetic attachment of labels, and the proposed methods have not been commercially viable alternatives to standard synthesis approaches (10-12).

Recently Prof. Brown’s group discovered that the neutral heteroaromatic “click” backbone, when it introduced instead of natural phosphodiester bond is acceptable for Taq polymerase and can be used for most polymerase dependent proceses (13).

Cooper dependent “click chemistry” often limits that type of attachment chemistry due to cytotoxicity. Recently developed azadibenzocyclooctyne doesn’t require any catalysts and it is highly reactive towards aliphatic and aromatic azides (14).

Figure 2. Cooper free 1,3-dipolar cycloaddition.

Diels-Alder attachment method

Another important catalyst free chemo-selective attachment method is Diels-Alder reaction that was successfully employed in bio-conjugation.15 In order to make this process highly efficient at ambient temperature, the alkyldienyl group should be activated with electron donating group (EDG) and the dienophile should have adjacent electron withdrawing group (EWG).

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Figure 3. Diels-Alder reaction used in bio-conjugation.
 

Biosynthesis Inc. offers not only a wide variety of modified oligonucleotides but also conjugates of peptides, proteins and antibodies as well as their cutom conjugation. 

References:

1. BSI Bioconjugation

2. Jablonski E, Moomaw EW, Tullis RH, Ruth JL. Preparation of oligodeoxynucleotide-alkaline phosphatase conjugates and their use as hybridization probes. Nucleic Acids Res. 1986 Aug 11;14(15):6115-28. [PMC]

3. Kahl, J. D., & Greenberg, M. M. (1999). Solution-phase bioconjugate synthesis using protected oligonucleotides containing 3'-alkyl carboxylic acids. Journal of Organic Chemistry, 64(2), 507-510. [ACS] 

4. Kachalova, A. V., Zatsepin, T. S., Romanova, E. A., Stetsenko, D. A., Gait, M. J., & Oretskaya, T. S. (2000). Synthesis of Modified Nucleotide Building Blocks Containing Electrophilic Groups in the 2′-Position. Nucleosides, Nucleotides & Nucleic Acids, 19(10–12), 1693–1707. [NNN]

5. Prakash TP, Kawasaki AM, Lesnik EA, Owens SR, Manoharan M. 2'-O-[2-(amino)-2-oxoethyl] oligonucleotides. Org Lett. 2003 Feb 20;5(4):403-6. [ACS]

6. Podyminogin MA, Lukhtanov EA, Reed MW. Attachment of benzaldehyde-modified oligodeoxynucleotide probes to semicarbazide-coated glass. Nucleic Acids Res. 2001 Dec 15;29(24):5090-8. [PMC]

7. Raddatz S, Mueller-Ibeler J, Kluge J, Wäss L, Burdinski G, Havens JR, Onofrey TJ, Wang D, Schweitzer M; Hydrazide oligonucleotides: new chemical modification for chip array attachment and conjugation. Nucleic Acids Research, 01 Nov 2002, 30(21):4793-4802. [pdf]

8. Timofeev E, Kochetkova SV, Mirzabekov AD, Florentiev VL; Regioselective immobilization of short oligonucleotides to acrylic copolymer gels. Nucleic Acids Research, 01 Aug 1996, 24(16):3142-3148. [pdf]

9. Rostovtsev VV, Green LG, Fokin VV, Sharpless KB. A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective "ligation" of azides and terminal alkynes. Angew Chem Int Ed Engl. 2002 Jul 15;41(14):2596-9. 

10. Ustinov A.V., Dubnyakova V.V., Korshun V.A. A convenient “click chemistry” approach to perylene diimide-oligonucleotide conjugates. Tetrahedron. 2008;64:1467–1473. doi: 10.1016/j.tet.2007.11.048. [sciencedirect]

11. Agnew, B. et al., US Patent application 20080050731/A1.

12. Ming, X, Leonard, P, Heindle, D, and Seela, F.; Azide-alkyne "click" reaction performed on oligonucleotides with the universal nucleoside 7-octadiynyl-7-deaza-2'-deoxyinosine. Nucleic Acid Symposium Series No. 52, 471-472, 2008. [pdf]

13. El-Sagheer AH, Brown T. Click nucleic acid ligation: applications in biology and nanotechnology. Acc Chem Res. 2012 Aug 21;45(8):1258-67. [PMC]

14. Debets MF, van Berkel SS, Schoffelen S, Rutjes FP, van Hest JC, van Delft FL. Aza-dibenzocyclooctynes for fast and efficient enzyme PEGylation via copper-free (3+2) cycloaddition. Chem Commun (Camb). 2010 Jan 7;46(1):97-9. [ChemComm]

15. Marchan V., Ortega S., Pulido D., Pedroso E., Grandas A. Diels-Alder cycloadditions in water for the straightforward preparation of peptide–oligonucleotide conjugates. Nucleic Acids Res. 2006;34:e24.[PMC] [PubMed]