Chapter 7 – Complementary Experimental Tools  269

7.2.5  NUCLEIC ACID OLIGO INSERTS

Short sequences of nucleotide bases (~10 base pairs), known as oligonucleotides (or just

oligos), can be used to label specific sites on a DNA molecule. A DNA sequence can be

cut at specific locations by enzymes called “restriction endonucleases,” which enables short

sequences of DNA complementary to a specific oligo sequence to be inserted at that loca­

tion. Incubation with the oligo will then result in binding to the complementary sequence.

This is useful since oligos can be modified to be bound to a variety of chemical groups,

including biotin, azide, and alkynes, to facilitate conjugation to another biomolecule or struc­

ture. Also, oligos can be derivatized with a fluorescent dye label either directly or via, for

example, a bound biotin molecule, to enable fluorescence imaging visualization of specific

DNA sequence locations.

7.2.6  APTAMERS

Aptamers are short sequences of either nucleotides or amino acids that bind to a specific

region of a target biomolecule. These peptides and RNA-​ or DNA-​based oligonucleotides

have a molecular weight that is relatively low at ~8–​25 kDa compared to antibodies that are

an order of magnitude greater. Most aptamers are unnatural in being chemically synthesized

structures, though some natural aptamers do exist, for example, a class of RNA structures

known as riboswitches (a riboswitch is an interesting component of some mRNA molecules

that can alter the activity of proteins that are involved in manufacturing the mRNA and so

regulate their own activity).

Aptamers fold into specific 3D shapes to fit tightly to specific structural motifs for a range

of different biomolecules with a very low unbinding rate measured as an equivalent dissoci­

ation constant in the pico-​ to nanomolar range. They operate solely via a structural recogni­

tion process, that is, no chemical bonding is involved. This is a similar process to that of an

antigen–​antibody reaction, and thus aptamers are also referred to as chemical antibodies.

Due to their relatively small size, aptamers offer some advantages over protein-​based anti­

bodies. For example, they can penetrate tissues faster. Also, aptamers in general do not evoke

a significant immune response in the human body (they are described as nonimmunogenic).

They are also relatively stable to heat, in that their tertiary and secondary structures can be

denatured at temperatures as high as 95°C but will then reversibly fold back into their original

3D conformation once the temperature is lowered to ~50°C or less, compared to antibodies

that would irreversibly denature. This enables faster chemical reaction rates during incuba­

tion stages, for example, when labeling aptamers with fluorophore dye tags.

Aptamers can recognize a wide range of targets including small biomolecules such as

ATP, ions, proteins, and sugars, but will also bind specifically to larger length scale biological

matter, such as cells and viruses. The standard method of aptamer manufacture is known

as systematic evolution of ligands by exponential enrichment. It involves repeated binding,

selection, and then amplification of aptamers from an initial library of as many as ~10¹⁸

random sequences that, perhaps surprisingly, can home in on an ideal aptamer sequence in a

relatively cost-​effective manner.

Aptamers have significant potential for use as drugs, for example, to block the activity of

a range of biomolecules. Also, they have been used in biophysical applications as markers

of a range of biomolecules. For example, although protein metabolites can be labeled using

fluorescent proteins, this is not true for nonprotein biomolecules. However, aptamers can

enable such biomolecules to be labeled, for example, if chemically tagged with a fluorophore

they can report on the spatial localization of ATP accurately in live cells using fluorescence

microscopy techniques, which is difficult to quantify using other methods.

A promising recent application of aptamers is in the fluorescent labeling of RNA in

living cells. To date, the best labeling technology for RNA in situ has been antibodies, using

RNA FISH and smFISH (see section 7.2.3). However, a drawback with both is the size of

the antibodies (Stokes radius ~10 nm) impairing functional activity of the RNA, also, for

smFISH that the experiments need to be performed on chemically fixed (i.e., dead) cells.

KEY BIOLOGICAL

APPLICATIONS:

BIOCONJUGATION

TECHNIQUES

Attaching biophysical probes;

Molecular separation; Molecular

manipulation.