268 7.2  Bioconjugation

The stalk of the Y structure is called the Fc region whose sequence and structure are rea­

sonably constant across a given species of animal. The tips of the Y comprise two Fab regions

whose sequence and structure are highly variable and act as a unique binding site for a spe­

cific region of a target biomolecule (known as an antigen), with the specific binding site of the

antigen called the “epitope.” This makes antibodies particularly useful for specific biomolecule

conjugation. Antibodies can also be classed as monoclonal (derived from identical immune

cells and therefore binding to a single epitope of a given antigen) or polyclonal (derived from

multiple immune cells against one antigen, therefore containing a mixture of antibodies that

will potentially target different epitopes of the same antigen).

The antibody–​antigen interaction is primarily due to significantly high van der Waals

forces due to the tight-​fitting surface interfaces between the Fab binding pocket and the

antigen. Typical affinity values are not as high as strong covalent interactions with Kd values

of ~10⁻⁷ M being at the high end of the affinity range.

Fluorophores or EM gold labels, for example, can be attached to the Fc region of IgG

molecules and to isolated Fab regions that have been truncated from the native IgG structure

to enable specific labeling of biological structures. Secondary labeling can also be employed

(see Chapter 3); here a primary antibody binds to its antigen (e.g., a protein on the cell

membrane surface of a specific cell type) while a secondary antibody, whose Fc region has

a bound label, specifically binds to the Fc region of the primary antibody. The advantage of

this method is primarily one of cost since a secondary antibody will bind the Fc region of all

primary antibodies from the same species and so circumvents the need to generate multiple

different labeled primary antibodies.

Antibodies are also used significantly in single-​molecule manipulation experiments. For

example, single-​molecule magnetic and optical tweezer experiments on DNA often utilize a

label called “digoxigenin” (DIG). DIG is a steroid found exclusively in the flowers and leaves

of the plants of the Digitalis genus, highly toxic to animals and perhaps as a result through

evolution has highly immunogenic properties (meaning it has a high ability to provoke an

immune response, thus provoking the production of several specific antibodies to bind to

DIG), and antibodies with specificity against DIG (called generally “anti-​DIG”) have very high

affinity. DIG is often added to one end of a DNA molecule, while a trapped bead that has been

coated in anti-​DIG molecule can then bind to it to enable single-​molecule manipulation of

the DNA.

DIG is an example of a class of chemical called “haptans.” These are the most common sec­

ondary labeling molecule for immuno-​hybridization chemistry due to their highly immuno­

genic properties (e.g., biotin is a haptan). DIG is also commonly used in fluorescence in situ

hybridization (FISH) assays. In FISH, DIG is normally covalently bound to a specific nucleo­

tide triphosphate probe, and the fluorescently labeled IgG secondary antibody anti-​DIG is

subsequently used to probe for its location on the chromosome, thus allowing specific DNA

sequences, and genes, to be identified following fluorescence microscopy.

A powerful application of FISH is for RNA imaging. RNA FISH can be used to visu­

alize specific mRNA transcripts in living cells and in tissue sections. The state-​of-​the-​art is

smFISH, which can enable single-​molecule detection on RNA transcripts of chemically fixed

cell samples.

7.2.4  “CLICK” CHEMISTRY

Click chemistry is the general term that describes chemical synthesis by joining small-​

molecule units together both quickly and reliably, which is ideally modular and has high

yield. It is not a single specific chemical reaction. However, one of the most popular examples

of click chemistry is the azide–​alkyne Huisgen cycloaddition. This chemical reaction uses

copper as a catalyst and results in a highly selective and strong covalent bond formed between

azide (triple bonded N–​N atoms) and alkyne (triple bonded C–​C bonds) chemical groups to

form stable 1,2,3-​triazoles. This method of chemical conjugation is rapidly becoming popular

in part due to its specific use in conjunction with increased development of oligonucleotide

labeling.