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Sunday, January 19, 2014

Aptamers - A new class of Oligonucleotides for Therapeutic and Diagnostic Use

Aptamers are oligonucleotides (DNA or RNA) having high affinity and specificity in identifying the target molecules. The word aptamer is derived from the latin “aptus – fit” and greek “meros – part”.

Aptamers are single-stranded RNA or DNA oligonucleotides 15 to 60 base in length that bind with high affinity to specific molecular targets; most aptamers to proteins bind with Kds (equilibrium constant) in the range of 1 pM to 1 nM similar to monoclonal antibodies. These nucleic acid ligands bind to nucleic acid, proteins, small organic compounds, and even entire organisms. Aptamers have many potential uses in intracellular processes studies, medicine and technology. Aptamers are often identified using a technique called SELEX (Systematic Evolution of Ligands by EXponential enrichment). By this techniques aptamers(oligos) having high affinity and specificity to the target is isolated from the sequence pool after several rounds of selection.

Aptamers have similar affinities as antibodies to the target molecules and provide various advantages, which includes greater stability, easier large scale production, and low immunogenicity.

Aptamer Structure

DNA Aptamers are of 15 - 60 oligonucleotide bases long.

Interaction of aptamers to the target is based on the three Dimensional folding patterns. The complex 3 Dimensional structure of the single stranded oligonucleotide is due to the intramolecular hybridization, which causes the folding into particular shape. Due to this complex three dimensional shape, aptamers have high affinity and specificity towards the target molecules.

Aptamer Selection Process : Invitro Selection
Theoretically it is possible to select aptamers virtually against any molecular target; aptamers have been selected for small molecules, peptides, proteins as well as viruses and bacteria.

Aptamer Selection Method
The aptamers are selected by incubating the target molecule in a large pool of oligonucleotide (usually 40 to 60mers), the pool size of the oligonucleotide is from 10^10 to 10^20. The large pool size of the oligonucleotide ensures the selection and isolation of the specific aptamer. The structural and informational complexity of the oligonucleotide pool and its functional activity is an interesting and active area to develop an algorithm based development of nucleic acid ligands. Aptamers can distinguish between closely related but non-identical members of a protein family, or between different functional or conformational states of the same protein. In a striking example of specificity, an aptamer to the small molecule theophylline (1,3-dimethylxanthine) binds with 10,000-fold lower affinity to caffeine (1,3,7-trimethylxanthine) that differs from theophylline by a single methyl group. The protocol called systematic evolution of ligands by exponential enrichment (SELEX) is generally used with modification and variations for the selection of specific aptamers. Using this process, it is possible to develop new aptamers in as little as two weeks.

Different Aptamer Selection Methods
  • Nitrocellulose Membrane Filtration-Based SELEX 
  • Affinity Chromatography and Magnetic Bead-Based SELEX
SELEX for small molecules, as well as proteins, can be carried out using this method. Several aptamers were selected using this method by making an affinity column containing target-immobilized beads. However, the disadvantage of this method is that it cannot be applied if the target lacks the affinity tag or the functional group needed for coupling to the beads.
  • Capillary Electrophoresis-Based SELEX
With the use of Capillary Electrophoresis-Based SELEX an aptamer can be selected by a mobility shift
among the mixture of a target, the library, and the target-library complex. In particular, the
greatest virtue of applying this method to SELEX is that the successful selection of the aptamer can be
achieved within very few rounds, generally 2–4 rounds, compared to other methods.
  • Microfluidic-Based SELEX
Microfluidic Based SELEX method is mainly processed on a chip, it is able to enhance selection efficiency on a small scale. For example, the DNA aptamer-specific bound to neurotoxin type B wasobtained after a single round of selection using the Continuous-flow Magnetic Activated Chip-based Separation (CMACS) device.
  • Cell-SELEX
Cell-SELEX is aimed at searching for an aptamer against a whole cell, whereas the primary targets of
other SELEX methods are single highly-purified proteins, where as the targets of Cell-SELEX
are extracellular proteins on the cell surface or unique structures of the cell.
  • Other Method-Based SELEX
AFM, electrophoretic mobility shift assays (EMSA), and surface plasmon resonance (SPR), have been performed in connection with SELEX.

Aptamers Advantages:

While aptamers are analogous to antibodies in their range of target recognition and variety of applications, they possess several key advantages over their protein counterparts:
  • Easier and more economical to produce. Aptamers are made through chemical synthesis, a process that is highly reproducible and can be readily scaled up. Their production does not depend on bacteria, cell cultures or animals.
  • Compared to antibodies, toxicity and low immunogenicity of particular antigens do not interfere with the aptamer selection.
  • Aptamers are capable of greater specificity and affinity than antibodies.
  • Aptamers can easily be modified chemically to yield improved, custom tailored properties.
  • Aptamers can specifically bound to either small molecules and complex multimeric structures.
  • Small size of aptamers leads to a high number of moles of target bound per gram, and they may have improved transport properties allowing cell specific targeting and improved tissue penetration.
  • Aptamers are much more stable at ambient temperature than antibodies yielding a much higher shelf life, and they can tolerate transportation without any special requirements for cooling, eliminating the need for a continuous cold chain.
  • Ability to inactivate proteins, without altering genetic material.
Aptamers Disadavantages
  • Lower levels of affinities than antibodies.
  • Aptamers will not bind to some target molecules.
  • Aptamers identification is expensive and labor intensive.
Applications of Aptamers:

Aptamers has got various application in the following fields:
  1. New Drug Development
  2. Bio Imaging
  3. As Therapeutic Tool
  4. Drug Discovery
  5. Disease Diagnosis
  6. Hazard Detection
  7. Food Inspection

Review Articles: 

Aptamer-modified nanoparticles and their use in cancer diagnostics and treatment, Christine Reinemann, Beate Strehlitz- UFZ - Helmholtz Centre for Environmental Research, Leipzig, Germany.

Aptamers and Their Biological Applications, Kyung-Mi Song, Seonghwan Lee and Changill Ban - Department of Chemistry, Pohang University of Science and Technology, San31, Hyoja-dong, Pohang,
Gyeongbuk 790-784, Korea

Technical Resources -
 Base Pair Biotechnologies
Gene Link
Integrated DNA Technologies
NeoVentures Biotechnology Inc

Saturday, January 11, 2014

Estimation of cellulose

Cellulose is most abundant in nature and it is a major structural polysaccharide in plants. Cellulose is composed of linear chain of glucose units which is linked together by beta glycosidic bonds.


Cellulose undergoes acetolysis with acetic/nitric reagent forming acetylated cellodextrins wich gets dissolved and hydrolyzed to form glucose molecules on treatment with 67% H2SO4. This glucose molecule is dehydrated to form hydroxymethyl furfural which forms green colored product with anthrone and the color intensity is measured at 630nm.

  • Acetic/Nitric Reagent: Mix 150ml of 80% acetic acid and 15mL of concentrated nitric acid.
  • Anthrone: Dissolve 200mg anthrone in 100mL of ice-cold 95% sulphuric acid. Prepare fresh and chill for 2h before use.
  • 67% sulphuric acid
  1. Add 3mL acetic/nitric reagent to a known amount (0.5g or 1g) of the sample in a test tube and mix in a vortex mixture.
  2. Place the tube in a water bath at 100°C for 30min.
  3. Cool and then centrifuge the contents for 15-20min.
  4. Discard the supernatant.
  5. Wash the residue with distilled water.
  6. Add 10mL of 67% sulphuric acid and allow it to stand for 1h.
  7. Dilute 1mL of the above solution to 100mL.
  8. To 1mL of this diluted solution, add 10mL of anthrone reagent and mix well.
  9. Heat the tubes in boiling water bath for 10min.
  10. Cool and measure the color at 630nm.
  11. Set a blank with anthrone reagent and distilled water.
  12. Take 100mg cellulose in a test tube and proceed from step No. 6 for standard. Instead of just taking 1mL of the diluted solution (Step 7) take a series of volumes (say 0.4 to 2mL corresponding to 40-200mg of cellulose) and develop the color.

1. Updegroff, D M (1969) Anal Biochem.

Thursday, January 9, 2014

Avidin Biotin System

Avidin - Biotin systems are widely used in biochemical analysis and has got various applications in biotechnology and clinical medicine. The avidin-biotin system utilizes biotin, a low molecular weight vitamin, which can be chemically coupled to low or high molecular weight molecules such Protein, DNA or hormones. The biotin in its native or derivatized (having reporter probes ) form can be recognized by Avidin or streptavidin .

Avidin is a protein found in the egg white and streptavidin is found in stremptomyces avidinii. Both Avidin and Streptavidin has very high binding affinity towards the biotin.

Biotin is a low molecular weight vitamin commonly known as the vitamin H. Biotin has various roles in human body, first of all its an essential nutrient, biotin is also involved in the biosynthesis of fatty acids, gluconeogenesis, energy production and de novo synthesis of purines nucleotides.

Structure of Biotin

Structure of Biotin / Vitamin H

Biotin (C10H16N2O3S) has a Molecular Weight = 244.31 and is composed of
  • An ureido ring fused with a tetrahydrothiophene ring,
  • A valeric acid substituent attached to 1 of the 2 carbon atoms of the tetrahydrothiophene ring.
Biotin - Avidin Based Detetction System

Probes used in the Avidin-Biotin System

Probes Used can be prepared in two ways:

1. Chemically Coupled Probes
2. Biotinylated Probes

Chemically Coupled Probe
Avidin or streptavidin can be chemically coupled and are fluorescent, radioactive, or other types of macromolecules (proteins,polysaccharides, etc.).

Biotinylated ProbesOther method is to Biotinylate the probes and to make it interact them with streptavidin under sub-saturating ratios, thus leaving extra binding sites vacant.

More recently, fusion proteins have been prepared of streptavidin with different enzymes and native fluorescent proteins.

Which is best to use Avidin or Streptavidin?
Streptavidin is generally preffered over Avidin, because of its of neutral charge and lack of glycosylation. Nowadays various derivatives of avidin are available which are less expensive.

Types of Probes Used in the Avidin-Biotin System
  • Enzymes 
  • Radiolabels 
  • Flourescent Agents 
  • Chemilumniscent Agents 
  • Chromophores 
  • Heavy Metals 
  • Colloidal Gold 
  • Ferritin 
  • Hemocyanin 
  • Phages 
  • Macromolecular Carriers 
  • Liposomes 
  • Solid Support 
Applications of Avidin - Biotin System
  • Isolation Studies
  • Affinity Chromatography
  • Affinity Precipitation
  • Immobilizing Agents
  • Enzyme Reactor Systems
  • Selective retrieval
  • Selective Elimination
  • Diagnostics
  • Immunoassay
  • Signal Amplification
  • Blotting Technology
  • Bioaffinity Sensor
  • Gene probes
  • Chromosome Mapping
  • Affinity Cytochemistry
  • Localization Studies
  • Light Microscopy
  • Flourescence microscopy
  • Electron Microscopy
  • Histochemistry
  • Phage-display technology
  • Hybridoma Technology
  • Epitope Mapping
  • Cell Separation
  • Flow Cytometry
  • Fusogenic agent
  • Monolayer Technology
Advantages and Disadvantages of Avidin and Streptavidin
Advantage of Avidin is its ability of reversible interaction, in many applications reversible interaction is desired. Streptavidin has strong binding to Biotin which cannot be reserved can be disadvantageous. Avidin monomers can be immobilized and can be used for reversible interactions.

Encyclopedia in Molecular Biology