Functionalizing Morpholino Oligos for Antisense Drug Research and Development

Special Article - Antisense Drug Research and Development

J Drug Discov Develop and Deliv. 2016; 3(1): 1021.

Functionalizing Morpholino Oligos for Antisense Drug Research and Development

Li Yong-Fu*

Gene Tools, LLC, USA

*Corresponding author: Li Yong-Fu, Gene Tools, LLC, 1001 Summerton Way, Philomath, OR97370, USA

Received: January 28, 2016; Accepted: April 27, 2016; Published: May 02, 2016


A Morpholino oligo itself is difficult to physically detect in a biological assay system, and is also poorly cell-permeable in cultures and living animals. This article provides an overview of the state of the art for solving these problems using corresponding functionalizations. Modification at the ends of a Morpholino oligo gives functional groups or functional entities; the former are useful for further conjugation with custom-designed molecules, whereas the latter may provide fluorophores for optical detection or delivery-enabling moieties for in vivo antisense activity studies. The combination of both ends, in particular with double functionalization at the 3’-end, yields myriad opportunities for more diverse applications. In addition, a photo-cleavable linker inserted into a Morpholino oligo enables control of gene expression in a spatially and temporally controllable manner.

Keywords: Morpholino oligo; Functionalization; End-modification; Conjugation


RNA: Ribonucleic Acid; mRNA: messenger RNA; siRNA: small interfering RNA; PNA: Peptide Nucleic Acid; S-DNA: Phosphorothioate-Linked Deoxyribonucleic Acid; LNA: Locked Nucleic Acid; UV: Ultraviolet


Drug discovery is arriving at an advanced stage where drug design and development is becoming simpler, made possible through antisense molecules. This advancement arises from the small number of bases of oligonucleotides involved in the hydrogen-bonded bridges of Watson-Crick base pairing, giving predictable interactions with their complementary strands of nucleic acids. As advances in oligo structure have improved their efficacy, specificity and non-toxicity, antisense technology has thus entered into drug research and development as drug candidates themselves. Antisense oligomers can bind and deactivate defective mRNAs when aberrant proteins are made in the body, or usefully alter the mRNAs to treat the genetic diseases when a required protein cannot be produced [1].

Morpholino oligos are among those antisense oligomers including siRNA, PNA, S-DNA, and LNA, but stand out as superior [2] for several reasons: (1) they are free of off-target effects because their uncharged backbone does not interact electrostatically with proteins; (2) each is specific for a particular nucleic acid sequence because the oligo must bind about 14 contiguous bases or more to significantly block a gene transcript, and (3) they are completely stable in biological systems because they are resistant to cleavage by nucleases [3]. However, like other types of antisense oligomers, Morpholino oligos themselves face two practical problems: (1) they are invisible by optical microscopy in biological assay systems, and (2) they cannot easily pass through the cell membrane to reach their intracellular targets.

The power and advantages of assessing intracellular processes at their most fundamental level has propelled the science of Morpholinos into bioconjugate chemistry where particular chemical groups are required to be created to effect coupling, to be modified to realize sensitive detection, or to be functionalized to get delivery in cell culture and animal studies. The success of conjugation schemes depends on the presence of the correct chemical groups. Every chemical modification or conjugation process involves the reaction of one functional group with another, resulting in the formation of a covalent bond. The creation of bioconjugate reagents with spontaneously reactive or selectively reactive functional groups forms the basis for simple and reproducible cross-linking of target molecules. Of the hundreds of reagent systems described in the literature or offered commercially, the most common chemical bond formations can be reduced to a couple dozen or so primary reactions. In this article, amide, carbamate, and triazine linkage are the most preferred derivatization strategies to functionalize Morpholino oligos. Primary and secondary amino groups and Click coupling components (alkyne and azide) are the most preferred functional groups for further conjugation.

This article is designed to provide a general overview of what is available for functionalizing Morpholino oligos including the reactive group installation, fluorophore attachment, cell-permeable moiety conjugation, and photo-switch assembly, all of which are illustrated with short descriptions of their properties and use along with a visual representation of the chemistry of bond formation. This is not meant to be an exhaustive discussion on the theory or mechanism behind each functionalization, nor is it a review of every application in which each functionalization has been used in chemistry or biology. On the last section of advanced modification, the main purpose is to open reader’s minds to what can be available and how to make the full use of the chemistry for developing multi-functional Morpholino oligos for unique or diverse applications.

Chemical Functionalization for Bioconjugation

Chemical attachment of a functional component to a Morpholino oligo forms the basis for constructing a reactive moiety. Unfortunately, the methods developed to cross-link other biological molecules such as proteins often do not apply to Morpholino oligos. The major reactive sites on proteins involve primary amines, sulfhydryls and others that are relatively easy to derivatize. Morpholino oligos contain none of these functional groups. There are particular sites that can be modified on the bases or the linkage between subunits to produce derivatives able to couple with a second molecule. However, modifications on these sites may impact the pairing of a Morpholino oligo with its complementary target sterically and/or electronically. Therefore, the ends of the Morpholino oligo have been the sites of choice for the introduction of functionalization.

5’-end OR 3’-end single modification

In the solid phase synthesis of Morpholino oligos, functionalization at the 5’-end of the oligo takes advantage of the tri-functional triazine moiety whereby installation of both the functional group and the site for oligo elongation can be accomplished. 3’-End modification uses the secondary amine of the last Morpholino subunit as a reactive site for functionalization.

Amino groups are probably the most versatile functional moiety for post-synthetic derivatization. They are reactive towards isothiocyanate, isocyanate, acylazide, activated ester, sulfonyl chloride, aldehyde, epoxide, carbonate, arylating agent, imidoester, and anhydride. Among those, the acylation of amine with activated ester is rapid and occurs in high yield to give a stable amide bond. The primary amine can be installed at the 5’-end (5A, Figure 1), or 3’-end (3A, Figure 2).