Notes on Nucleic Acid Bioconjugation

Credit for Images: Bioconjugate Techniques (Greg T. Hermanson)

 Applications and Challenges of DNA Bioconjugates

• Modified oligonucleotides are used in a variety of techniques that involve hybridization.
• Sensitive hybridization reagents can use oligonucleotides that are labeled with a fluorescent marker or an enzyme that produces observable products.
• DNA does not include many of the easily modified functional groups present in proteins.
• To modify DNA, other functional groups usually must be attached first.
• The two main categories of DNA bioconjugation techniques are chemical and enzymatic. Some enzymatic approaches are described here.

Random-Primed Labeling

• Random-primed labeling is a form of enzymatic DNA bioconjugation that uses a reaction process with just a single denaturation step, a single annealing step, and a single extension step.
• The following materials are added to a mixture for random-primed labeling.
  • Random hexa-deoxynucleotides to serve as primers.
  • dNTPs (ordinary nucleotides).
  • Modified dNTPs such as ³²P radiolabeled nucleotides (which do not affect the probe’s hybridization efficiency), fluorescent dye labeled nucleotides, or affinity molecule labeled nucleotides (which bind observable compounds).
  • Template DNA.
  • The Klenow fragment version of DNA polymerase I that lacks 5’-3’ exonuclease activity.
• The ratio of ordinary dNTPs to modified dNTPs decides the magnitude of labeling by determining the amount of modified dNTPs that are incorporated.
• Random-primed labeling produces a random array of oligonucleotides of different lengths and sequences corresponding to different parts of the template.
  • This is because the oligonucleotides are generated by the replicated sequences between annealed random hexa-deoxynucleotide primers (the oligonucleotides also include the primer sequence at their 5’ ends).

Nick Translation Labeling

• Nick translation labeling is a form of enzymatic DNA bioconjugation that uses a 15°C incubation temperature for the reaction.
• The following materials are added to a mixture for random-primed labeling.
  • dNTPs (ordinary nucleotides).
  • Modified dNTPs such as affinity tagged nucleotides or fluorescently tagged nucleotides.
  • Template DNA.
  • Mg²⁺ and low concentrations of pancreatic DNase I (which only has single-stranded endonuclease activity in the presence of Mg²⁺).
  • DNA polymerase I.
• The small amount of DNase I with Mg²⁺ makes a few single-stranded nicks in the template, allowing the DNA polymerase I to begin nick translation. In this way, new dNTPs and labeled dNTPs replace the nucleotides in the original template molecule.
• The ratio of ordinary dNTPs to modified dNTPs decides the magnitude of labeling by determining the amount of modified dNTPs that are incorporated.

PCR Labeling

• PCR labeling is a form of enzymatic DNA bioconjugation that uses a PCR reaction.
• The standard PCR components are added to the reaction. In addition, some labeled dNTPs or primers containing labeled nucleotides are also added.
• When labeled dNTPs are added, the ratio of ordinary dNTPs to modified dNTPs decides the magnitude of labeling by determining the amount of modified dNTPs that are incorporated.
• RT-PCR versions of PCR labeling can be used to create sensitive assays for RNA viruses.

Terminal Transferase Labeling

• Terminal transferase labeling uses terminal deoxynucleotidyl transferase (TdT) to add a labeled dNTP to the 3’ ends of DNA oligonucleotides without the need for a template.
• TdT (found in certain lymphocytes) usually adds multiple non-templated nucleotides to the 3’ ends of oligonucleotide strands.
• In the presence of Co²⁺ salts, TdT only adds single non-templated nucleotides to the 3’ ends.
• By applying TdT to add a labeled dNTP to the 3’ ends of oligonucleotides, the middle of the oligonucleotide strand will not have its hybridization efficiency disturbed. This is especially useful for large labels like biotin-streptavidin detection conjugates or many fluorescent dyes.

T4 RNA Ligase 1 Labeling

• T4 RNA ligase 1 labeling uses a very similar mechanism to that of TdT labeling. T4 RNA ligase 1 adds a single nucleotide to the end of an RNA oligonucleotide.
• This also has the benefit of not affecting hybridization efficiency.
• Unlike the other methods, T4 RNA ligase 1 labeling requires labeled bis-phosphoryl nucleotide derivatives (which have one phosphate on the 5’ end end one phosphate on the 3’ end) rather than labeled dNTPs.

Individual Labeled Nucleotides

• Although many modifications can be made to individual nucleotides, most block the activity of enzymes used for labeling of oligonucleotides. However, some will allow the activity of one or more type of labeling enzyme (see diagram).
• Sometimes, it can be beneficial to add an amine to a nucleotide so that the amine can react with other chosen molecules to form various types of conjugates.
• There are numerous chemical techniques for labeling individual nucleotides.