DNA Bioconjugation Methods and Techniques

Deoxyribonucleic acid (DNA) is the fundamental molecule that carries the genetic code of all known life. It is formed of two polynucleotide chains coiled around each other in a double helix arrangement. One fascinating use for DNA is in DNA bioconjugates which can be used in a variety of applications such as biosensors, bioimaging markers, drug delivery, and more. In this article, we discuss DNA bioconjugation methods and techniques with examples from industrial and research applications.

DNA bioconjugation methods and techniques include non covalent conjugation and covalent conjugation using a variety of functional groups including aldehydes, azides, thiols, and amines. 

DNA Chemical Structure

DNA is composed of two strands of polynucleotides arranged into a double helix by covalent interactions. Each chain is made up of a sugar-phosphate backbone. Each sugar is linked to a nucleotide. This can be one of four different nucleotides: adenine (A), thymine (T), guanine (G), and cytosine (C) which make up the fundamental genetic code. Each of these nucleotides can pair with their opposite nucleotide via hydrogen bonding: A to T and C to G. These hydrogen bonds hold the two strands of the double helix together. 

The structure of DNA is formed of two polynucleotide chains with a sugar-phosphate backbone arranged in a double helix. Bases are paired together A with T and C with G to create the genetic code. 

Structure of DNA
Structure of DNA (image source)

DNA Bioconjugation Chemistry

There are many different ways to prepare DNA for bioconjugation reactions. Since DNA has such a large chemical structure, it’s necessary to decide which part of the DNA structure needs to form the conjugate bond. For example, you can create conjugations at the end of the DNA chain, at the nucleotides, or as part of the phosphate backbone. 

DNA bioconjugation chemistry can be carried out at the termini, at nucleotides, or at part of the phosphate backbone. At these locations, DNA can form bioconjugates through non-covalent bonding, chemical functionalization, and functionalization during DNA synthesis. 

Non-Covalent Bonding with DNA

DNA can form conjugates without any modification using non-covalent interactions to form bonds to the other molecule. These bonds are often weak and are easily affected by the medium in which the bioconjugate is stored, particularly in terms of pH. For example, in this paper, the authors demonstrated the ability for DNA to be used to make covalent and non-covalent bioconjugates with gold nanoparticles. For more information on conjugation to PEGylated nanoparticles take a look at our article.


An interesting type of non-covalent bonding using DNA is intercalation. This is where a molecule is inserted between the bases of the DNA structure. Most commonly, this is the basis of certain types of poisoning which disrupt DNA. However, intercalation can also be used to create conjugates with various practical uses. For example, the authors of this paper were able to produce a fluorene intercalated DNA conjugate which has optical applications. Another example of intercalated DNA conjugates is found in this paper where the authors used DNA intercalated with oligoethylene-glycol-functionalized small molecules. This allowed them to produce a DNA conjugate-based thermoresponsive polymer.

DNA bioconjugation using intercalation
Intercalation of Ethidium between two DNA chains. Image from Wikipedia.

Chemical Functionalization of DNA Ends

DNA can also be used to form conjugates by performing chemical modifications to the DNA structure. For example, by functionalizing the ends of the DNA chain, the DNA can be conjugated to another molecule end to end. The authors of this review explore the many ways to functionalize DNA at the end of its chain, including solid-phase functionalization and solution coupling. Here are some examples of some of the types of couplings that can be used for producing DNA conjugates:

Amide Coupling

Amide bonds are excellent for conjugating DNA to other molecules. Amide chemistry is versatile and well-understood, opening up a range of options for conjugation reactions. For example, DNA-polymer conjugates can be produced using amide coupling reactions. In this paper, the authors covalently conjugated DNA to poly-isopropylacrylamide in organic solvents. Amide bonds were also used in the synthesis of DNA-quantum dot conjugates in this paper. Amine-DNA was attached to the CdSe/ZnS quantum dots using EDC and NHS chemistry.

Aldehyde-Based Methods

Aldehyde functional groups can also be used for DNA-conjugate synthesis. The authors of this paper introduced 4-formylbenzoic acid to the 5’-termino amino group of the nucleotides found in the structure of DNA. These groups were then used in a series of conjugation reactions for the purpose of producing novel assays for the detection of various kinases or proteases. The authors were able to validate their synthetic methodology by producing about three thousand DNA-peptide conjugates.

Thiol-Based Methods

Like hydroxyl groups, thiols can also be used to make effective conjugate bonds. For example, the authors of this paper used thiol-disulfide exchange reactions to conjugate DNA to proteins. The authors intended for their method to be used as a new tool for studying and manipulating proteins in biological systems. In this case, the non-native functional groups were added to the sugar-phosphate backbone of the DNA structure. We’ve covered other protein conjugation methods in a different article. We’ve also covered protocols used in cysteine bioconjugation techniques (which includes the thiol groups) in another article.

Thiols react readily and can be used for conjugation reactions. Papyrus Bio has a range of thiol-based conjugation kits. Explore thiol conjugation kits here.

Functionalization of During DNA Synthesis

Alternatively, DNA can also be functionalized for conjugation reactions by making adjustments to the structure during its synthesis. This can be achieved using dynamic chemistry to form the DNA from its building blocks, like in this paper, or by using enzymes to modify the DNA structure like in this paper.

Enzymatic methods

There is a significant amount of research into DNA modifying enzymes, particularly their practical application in terms of synthesizing new DNA conjugates. Even major scientific suppliers, such as Thermo Fisher, sell modifying enzymes that can be used to make modifications to DNA and RNA. The use of enzymes to produce base-modified DNA in particular can be used for detecting protein-DNA interactions, conjugating DNA with binding proteins, and modifying protein-DNA interactions (source). 

Site-specific Bioconjugation Within DNA

In order to achieve site-specific bioconjugation within DNA, you may need to make modifications to the bases that make up the internal structure of the double helix. This can be achieved by introducing modified bases into the DNA synthesis process. 

Site-specific conjugation within DNA can be achieved by integrating modified bases into the DNA structure during synthesis. Then, the functional groups on modified bases can be attached to other biomolecules using common chemistries such as alkyne azide cycloaddition (click chemistry).

Modified bases are found in nature and have fascinating effects on the properties of DNA. They contain different functional groups compared to the common DNA bases. Modified bases are also widely available from a range of suppliers such as Glen Research and IDT. By choosing the right bases with available functional groups, you can synthesize DNA that allows you to perform site-specific bioconjugation reactions using the functional groups available on the modified bases. You can also make further modifications to the nucleotides before you integrate them into the DNA structure, such as introducing protecting groups or alternate functional groups. 

However, DNA bioconjugation has a lot of challenges. Read about them more in our article.

Site-Specific Bioconjugation of DNA Using Alkyne and Azide Chemistry

Click chemistry using alkyne and azide bioconjugation chemistry is a well understood synthetic technique. It can be used for the site-specific bioconjugation of DNA to various other molecules. For example, in this paper, the authors successfully produced a covalent antibody-DNA conjugate for the detection of IgE and IgM antibodies. They specifically relied on a strain-promoted azide-alkyne cycloaddition reaction to achieve this conjugation. However, the IgE antibody did lose it’s specificity after the conjugation reaction. Besides click chemistry, there are other orthogonal bioconjugation techniques that we discuss in a separate article.

Trying to attach molecules together? You can explore conjugation kits to help you attach biomolecules together quickly and repeatably here.

Site-Specific Bioconjugation of DNA Using Thioether Bond Formation

The authors of this paper managed to bind a single long strand of DNA to an antibody using the formation of a thioether bond. Their reason for  this was because the site-specific conjugation of double-stranded DNA to an antibody enables the production of drug conjugates that also have DNA attached to them for barcoding purposes. However, their reaction was limited to a specific antibody which was able to tolerate increased temperatures. 

DNA Bioconjugation Applications

There are many applications for DNA bioconjugates that include fields such as genetics, materials sciences, biosensors, drug delivery, and more. 

DNA bioconjugation applications include attaching DNA to a protein C-terminus, coupling DNA to tyrosine residues in proteins, and bioconjugation of DNA to nanoparticles. 

The authors of this paper produced a site-specific conjugate of DNA with a protein at its C-terminus by using an expressed protein ligation technique. A range of homogenous DNA-protein conjugates were synthesized, all of which were bonded at the protein C-terminus. The authors relied on N-hydroxysuccinimide chemistry to conveniently couple the two molecules. The authors noted potential applications for their conjugate including the production of new assays using DNA-directed immobilization. 

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By attaching DNA to the C terminus of a protein it’s possible to add a barcode. In this paper, the authors used barcoded antibodies to determine what surface antigens were present on cancer cells. Image from Science.

Similarly, the authors of this paper used click chemistry to produce bifunctional linkers to produce protein-DNA conjugates through binding at the tyrosine residues. They achieved their synthesis of this conjugate using streptavidin and myoglobin. The bioconjugate produced was able to preserve the functionality of both the DNA and the protein, opening it up to many potential uses. The authors noted that their conjugate had higher enzymatic activity than the corresponding conjugate bonded at the Lysine residues. By being able to maintain the functionality of both the DNA and the protein, the authors believed that their method would be useful in the production of new protein based drugs, as well as potentially in biological imaging applications. However, the stability and relative ease of working with streptavidin and myoglobin does not guarantee that other proteins will retain their functionality when conjugated to DNA using this method. 

As mentioned previously, DNA has been comprehensively studied in combination with nanoparticles. DNA-nanoparticle conjugates have a range of applications thanks to the diverse potential of both DNA and nanoparticle chemistry. For example, the authors of this paper produced DNA functionalized gold nanoparticles which can be used to diagnose diseases like cancer and for targeted drug delivery to those diseased tissues. They used organosulfur anchor groups to attach the DNA to the gold nanoparticles and tested their stability. For alternative bioconjugation methods treating cancer, check out our article about bifunctional chelating agents.

Similarly, the authors of this paper used silver nanoparticles to produce DNA-silver nanoparticle conjugates that were used for the quantitative detection of HIV DNA. The authors reported a one-step incubation method that relied on thiol functionalized DNA molecules. The conjugates were used for this application because of their strong plasmon resonance scattering signals. 

DNA has also been attached to carbon nanotubes for barcoding, tracking, and therapeutic applications. Take a look at our article about this topic. We’ve also covered further details on bioconjugation to nanoparticles in a separate article.

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