Morpholinos are antisense molecules which are specific, stable, effective and nontoxic. They generally are comprised of about 25 nucleic acid bases linked by an uncharged synthetic backbone. They bind to complementary sequences of RNA by base pairing to prevent processes from happening at the bound sites. They are used in research to change the expression of proteins or to suppress the activity of noncoding RNA. Most commonly they are used in one of three ways:

• TRANSLATION BLOCKING: They can stop the progression of a ribosomal initiation complex toward the start codon of an mRNA, preventing translation of a protein.

• SPLICE MODIFICATION: They can interfere with splicing of pre-mRNA, changing the mRNA produced by the splicing process (usually by deletion of an exon).

• MICRO-RNA BLOCKING: They can suppress maturation of a primary miRNA by blocking the Drosha or Dicer cleavage sites, preventing the miRNA from reaching its mature and active form; alternatively, they can block the mRNA target of an miRNA.

Morpholinos are commonly used in zebrafish embryos, a system exquisitely sensitive to toxins and teratogens. They are also used in other embryonic systems, in cell cultures, in organ explants, and in intact adult organisms. Morpholinos have entered human clinical trials for a range of diseases. Morpholinos can block the replication of many kinds of viruses. Papers have reported successful knockdowns with Morpholinos in animals, protists, bacteria and plants.

Morpholinos are not recognized by cellular enzymes. The backbone of a Morpholino is sufficiently different from the backbone of a nucleic acid that the enzymes which recognize anionic nucleic acids will not interact with an uncharged Morpholino. Morpholinos are not degraded in cells or in organisms. Morpholinos do not bind to toll-like receptors or cause immune responses.

While bathing cells in Morpholinos can sometimes result in antisense activity, the concentrations needed are very high. In practical applications, one of several techniques must be used for delivering the Morpholinos to the cytosol of cells.

• For embryos Morpholinos are generally microinjected or electroporated.
• For cell cultures Morpholinos can be electroporated, scrape-loaded, or delivered with an endosomal escape reagent (e.g. Endo-Porter).
• For in vivo applications Morpholinos can be conjugated with arginine-rich cell-penetrating peptides or guanidinium dendrimers (e.g. Vivo-Morpholinos) for administration systemically by i.v. or i.p. or for localized administration by i.m., subcutaneous injection, intranasal administration or intracerebroventricular infusion.

As an experimental tool for manipulating cells or organisms at the RNA level, Morpholinos have been proven effective with many papers in the scientific literature describing their use (pubs.gene-tools.com). As a therapeutic, Morpholinos have shown great promise and established an excellent record of clinical safety (www.avibio.com). In my opinion, as existing techniques for cytosolic delivery of Morpholinos are improved and new techniques are developed, Morpholinos will be applied to new biological model systems in research and find their place as an accepted and powerful class of therapeutic compounds for humans.