ZNA™-Oligonucleotides Introduction

metabion is proud to offer a novel class of modified oligonucleotides improving hybridization properties of primers and probes.

 

Zip nucleic acids (ZNAs) are oligonucleotides conjugated with cationic spermine units that increase affinity for their target by decreasing electrostatic repulsion between negatively charged anionic single strand Nucleic Acids to improve hybridization, thus enhancing and accelerating target recognition.

The possibility of modulating the global charge of the ZNA oligonucleotide-oligocation conjugates by the number of cationic spermine moieties attached to the Nucleic Acid oligomer is key to easily predict melting temperature of ZNA-DNA/ZNA-RNA hybrids. Tm increases linearly with the length of the oligocation.

ZNAs were shown to enable specific and sensitive reactions when used as primers for PCR and Reverse Transcription. Moreover ZNA probes provide broad flexibility in assay design and represent an effective alternative to Minor Groove Binder (MGB)- and Locked Nucleic Acid (LNA)-containing oligonucleotides.

... featuring

• enhanced/accelerated target recognition
• increased sensitivity
• high specificity
• increased Tm for short probes
• improved quenching for long probes
• increased flexibility in terms of
  • efficiency at low oligo concentration
  • efficiency at high annealing temperatures
  • Mg²⁺ concentration adjustment
  • design/sequence composition constraints

... providing for

• efficient mismatch/SNP/allelic discrimination
• earlier C_t values
• improved quantification accuracy of low abundant transcripts
• improved RNA to cDNA conversion
• higher signal level

... hence qualifying for

      a wide range of Nucleic Acid-based applications like

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

Scan - Click - Zip and Stick – that´s the way it works …

 

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Analyzing and utilizing our one year´s experience with ZNA we are happy to extend and adjust our primer & probes portfolio for the benefit of increased design and thus application flexibility.

As widely and commonly known by now, adding Tm and/or hybridization sensitivity increasing modifications to oligonucleotides without losing specificity enables to compensate for sequence specific primers and/or probes design restrictions. ZNA-modified oligonucleotides have successfully proven to ease those restrictions .

The relative Tm increasing effect of ZNA or other duplex stabilizing groups is naturally higher on shorter oligonucleotides used as primers or probes. Hence, we have extended our ZNA length ranges now starting from 8mers for primers and 10mers for dual labeled probes!

 

Paying respect to the global charge of the ZNA oligonucleotide-oligocation conjugates raising solubility issues, we additionally offer ZNA-2 and ZNA-3 (cationic) building blocks for (anionic) primers ranging from 8 to 15mers, and dual labeled probes ranging from 10 to 17mers, respectively. Attachment of ZNA-4 and ZNA-5 building blocks to primers is allowed from 16mers (ZNA-4), and 20mers (ZNA-5), respectively. Attachment of ZNA-4 and ZNA-5 building blocks to dual labeled probes is allowed from 18mers (ZNA-4), and 22mers (ZNA-5), respectively.

Turning our experience in terms of physicochemical and application-oriented considerations into practice, maximum Nucleic Acid sequence length remains unchanged at 40 nucleotides for primers and probes, and maximum ZNA building block length is restricted to ZNA-5.

Additional affinity-to-target enhancement of primers and probes while maintaining specificity can be provided by incorporation of our base analogues 

C-5 propynyl-dC (pdC) raising melting temperature by ~2.8°C per substitution 

and/or

C-5 propynyl-dU (pdU) raising melting temperature by ~1.7°C per substitution.

 

On top of this, we are now also able to offer 5´ZNA modified dual labeled probes, introducing certain 5´reporters (i. e. FAM, Fluo, JOE,  ROX, TAMRA, CY3, CY5, CY5.5, …) through “click” chemistry. These probes are not degraded during PCR allowing for higher resolution post-PCR melt curve analysis if appropriately designed. Please inquire at info@metabion.com.  

Nota bene:  ZNA-building blocks can only be added 3´ or  5´.

All ZNA™ oligos will be delivered concentration adjusted (default 100 µM) and dissolved in H2O (pH 7-9).

 

Benefits from metabion´s extended ZNA™ -primers and probes portfolio:

• Increased design flexibility towards the use of shorter primers and probes for
  
  
• targeting highly conserved, specific (including A/T rich) sequences
• more sensitive mismatch discrimination providing improved mutation detection
• universal cycling conditions due to 'Tm leveling' effects (stabilization of A/T rich duplexes), hence supporting multiplex assays.
  
  
• Non cleavable (5´ ZNA-R) probes for post PCR melting curve analysis.
• Enhanced primer/probe sensitivity and specificity through incorporation of base analoga (pdC, pdU).

 

References and links

*1) Paris, C. et al. (2010) Zip nucleic acids are potent hydrolysis probes for quantitative PCR. Nucl. Acids Res., doi: 10.1093/nar/gkp1218. http://nar.oxfordjournals.org/cgi/content/abstract/gkp1218

*2) 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. http://nar.oxfordjournals.org/cgi/content/full/gkp661

*3) 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. http://cat.inist.fr/?aModele=afficheN&cpsidt=20744863
Download Article

*4) Voirin, E. et al. (2007) Versatile synthesis of oligodeoxyribonucleotide-oligospermine conjugates. Nat Protoc, 2, 1360-1367. http://www.nature.com/nprot/journal/v2/n6/full/nprot.2007.177.html
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5) ZNA™-FAQs