The concept to develop ZNA™ was to increase the affinity of oligonucleotides for their target by decreasing the electrostatic repulsions due to the polyanionic nature of nucleic acids. This was achieved by conjugating spermine derivatives as cationic moieties (Z units) to an oligonucleotide.

The ZNA™ synthesis is carried out using an oligonucleotide synthesizer following the standard phosphoramidite chemistry. The automated synthesis of ZNA™ allows any modifications such as dyes and quenchers to be included.

ZNA™ molecules are versatile as the number of Z units can be chosen and placed either internally or at the end (5’, 3’) of the oligonucleotide. By selecting the number of cationic units, the global charge of the ZNA™ molecule can be modulated.

The melting temperature (T_m) of ZNA™ increases significantly and linearly with the number of cationic units grafted on the oligonucleotide, providing a convenient means for fine tuning hybridization temperatures.

HIGHER AFFINITY, IMPROVED BINDING AND FASTER KINETICS

ZNA™ exhibit an exceptional high affinity for their nucleic acid target mainly because they accelerate hybridization by binding to its target with fast kinetics.

ADJUSTABLE AND PREDICTABLE T_m

The T_m increases linearly with the number of grafted Z units, independently of the base sequence of the oligonucleotide and of the conjugation site of the cationic Z units (3’ or 5’). The T_m increase per Z unit is only dependent on the length of the oligonucleotide.
The T_m of the ZNA is then easily predictable, using a simple mathematical relation depending on the intrinsic DNA oligonucleotide T_m, on the length N of the oligonucleotide and on the number z of cationic units T_m (ZNA) = T_m (DNA) + 36z/(N-3.2). (Noir et al., JACS 2008)

EASY TO DESIGN

The required T_m is easily achieved by conjugating the appropriate number of Z units at either side of the oligonucleotide. The oligonucleotide sequence and the location of the cationic units do not affect the T_m increase provided by the modification.

ZNA™ ARE SPECIFIC

ZNA™ display strict recognition selectivity. Similarly to standard oligonucleotides, ZNA™ specificity rely on optimized conditions including salt concentration, annealing temperature, and target and ZNA™ concentrations. Due to the higher affinity of ZNA™, these conditions may differ from those optimized for standard molecules.

ZNA™ performances have been successfully tested in PCR and RT-PCR as primers and probes (see below).

With their outstanding affinity and their high sensitivity, ZNA™ have a wide potential in Nucleic Acid-based technologies such as:

• PCR / RT-PCR
• Capture probes
• Northern Blot / Dot Blot
• Microarrays
• In Situ Hybridization (ISH)

ZNA™ as PCR primers:

• Great performance at high annealing temperatures
• Effective at very low concentrations
• Robust amplification in AT-rich regions (see graph)
• Efficient under short cycling conditions

ZNA™ as RT-PCR primers:

• Improved RNA to cDNA conversion (see graph)
• More accurate quantification of low-abundant transcripts

ZNA™ dual-labeled probes improve 5’ nuclease assay

• Facilitated design of 5’ nuclease assays
• Efficient short probes for improved discrimination
• Reduced background of long probes
• Earlier C_q values
• High hybridization-triggered fluorescence

Custom ZNA oligonucleotides will be available soon from Sigma-Aldrich and metabion.

Polyplus is looking for partners who wish to develop assays and applications using ZNA™ oligonucleotides in Molecular Biology and Molecular Diagnostics.

If you are interested in testing our ZNA™ in your assays, please feel free to contact Leila Guerlain, Strategic Marketing Manager.

LICENSING

ZNA™ technology is available for licensing.

Polyplus is seeking to license synthesis and commercialization of ZNA™ molecules to oligonucleotide manufacturers. Polyplus is also looking for licensing ZNA™ to Molecular Biology or Molecular Diagnostics companies wishing developing and using ZNA™ in new applications.

If you are interested and if you would like to inquire about the licensing opportunities, simply contact Leila Guerlain, Strategic Marketing Manager.

REFERENCES

Paris, C. et al. (2010) Zip nucleic acids are potent hydrolysis probes for quantitative PCR. Nucl. Acids Res., doi: 10.1093/nar/gkp1218. Download the PDF

Moreau et al. (2009) Zip nucleic acids (ZNAs): new high affinity oligonucleotides as potent primers for PCR and reverse transcription. Nucl. Acids Res., 37: e130; doi:10.1093/nar/gkp661. Download the PDF

Noir, R. et al. (2008) Oligonucleotide-oligospermine conjugates (Zip Nucleic Acids): a convenient means of finely tuning hybridization temperatures. J Am Chem Soc, 130, 13500-13505.

Voirin, E. et al. (2007) Versatile synthesis of oligodeoxyribonucleotide-oligospermine conjugates. Nat Protoc, 2, 1360-1367.

INTELLECTUAL PROPERTY

Polyplus-transfection is the worldwide exclusive licensee of the patent “Cationic oligonucleotides, automated methods for preparing same and their uses” WO/2007/069092 A2 filed by the Centre National de la Recherche Scientifique (CNRS), December 15, 2005.
Polyplus-transfection shares with the CNRS and the Université de Strasbourg rights over the patent “Methods for hybridizing nucleic acids” WO/2009/083763 filed Dec 27, 2007.