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Oligo Modifications for Increased Duplex Stability & Nuclease Resistance

 
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02/08/2009

Oligo Modifications for Increased Duplex Stability & Nuclease Resistance

Oligo Modifications for Increased Duplex Stability & Nuclease Resistance

Antisense research requires short oligonucleotides that are complementary in sequence, and upon specific hybridization to its cognate gene product, induce inhibition of gene expression Increased stability of the RNA-DNA duplex in terms of hybridization and half-life and nuclease resistance are underlying requirements for successful gene inhibition. These modifications can also be used for molecular probes and primers. Listed are some of the common modifications that impart these properties.

 

Modification

Chemical Characteristic

Duplex Stability Nuclease Resistance

Locked Nucleic

Acids (LNAs)

Bicyclic nucleic acid where a ribonucleoside is linked between the 2'-oxygen and the 4'-carbon atoms with a methylene unit.

Duplex Stability: Highest thermal stability of all available modifications
Nuclease Resistance: Increased

Unlocked Nucleic

Acids (UNA’s)

The C2’ and C3’ atoms of the sugar moiety have been cleaved.

Duplex Stability: Enables fine tuning of duplex thermodynamic stabilities
Nuclease Resistance: Good

Morpholinos (MOs)

Sugar and diester linkages. Morpholinos replace the sugar part of the nucleotide for a morpholine rings (6 atom ring) instead of deoxyribose rings and linked through phosphor-diamidate groups instead of phosphates, thus rendering these molecules uncharged at physiological pH

Duplex Stability: Good
Nuclease Resistance: Good

Peptide Nucleic

Acid (PNAs)

The backbone is made from repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. The different bases (purines and pyrimidines) are linked to the backbone by methylene carbonyl linkages. Unlike DNA or other DNA analogs, PNAs do not contain any pentose sugar moieties or phosphate groups.

Duplex Stability: Strong binding and increase duplex stability
Nuclease Resistance: Good

Phosphorothioate

(PS)

Modification of the phosphor-diester bond by replacing one of the non-bridging oxygens by sulfur

Duplex Stability: Hybridizes to the target sequences with lesser affinity than oligos with phosphodiester backbone
Nuclease Resistance: Imparts resistance to nuclease degradation

Phosphonoacetate

Phosphate in which one of the oxygen atoms has been replaced with a carboxylic acid.

Duplex Stability: Good
Nuclease Resistance: Good

Propyne analogs

C-5 propyne analogs of dC and dT

Duplex Stability: Increased binding affinity to the target mRNA and increased stability
Nuclease Resistance: Increased nuclease resistance

2'-O-methyl RNA

2'-O-methyl at the 2’ hydroxyl position

Duplex Stability: Binding similar to DNA
Nuclease Resistance: Increased

5-Me-dC

C-5 methylated dC

Duplex Stability: Increased
Nuclease Resistance: Similar to DNA

2'-5' Linked

2'-5' linked phosphodiester linage, 3’ deoxy bases

Duplex Stability: Increased binding efficiency to RNA
Nuclease Resistance: Increased

Chimeric Linkages

Mixed phosphorothioate and phosphodiester linkages and modifications

Duplex Stability: Increased
Nuclease Resistance: Increased

Bioconjugation

conjugation with lipid and peptides

Improve stability and cellular uptake

 

Applications and Recommended Modifications:

Antisense Gene Target

  • 2'-O-Me-nucleotides (2'-O-Me-RNA) form more stable hybrids with complementary RNA strands than equivalent DNA and RNA sequences.
  • Phosphorothioate linkages confer resistance to nuclease degradation.
  • Locked Nucleic Acids (LNA) demonstrate unsurpassed duplex stability. Use phosphorothioate linkages to impart nuclease resistance and LNA bases to achieve the most stable hybridization.
  • Propyne modified with phosphorothioate linkages are 50x more effective than the corresponding phosphodiester oligo.
  • Morpholinos have high mRNA binding affinity and good specificity; they are free of off target expression modulation and do not induce innate immune responses.
  • Phosphonoacetate active in siRNA duplexes and accelerate the initial rate of cleavage by RNase H-1 when incorporated with phosphorothioates. Excellent penetration ability penetration of cultured cells.
  • Unlocked Nucleic Acids (UNA) decrease Tm of 5-10 0C per UNA monomer; aids in fine tuning of duplex thermodynamic stabilities and improve gene silencing
  • In compare PNA with BNA, bridged nucleic acid (BNA) has stronger binding properties and biological stability of BNA imply that a small quantity of BNA can be effective for therapeutic applications. Triplex invasion of a BNA shows good potential as antigene material. As a third generation molecule in antisense therapy, there has been experimental data that shows good effect in vitro and in vivo.

Real-Time PCR probes and QPCR

  • 5-Me-dC enhances duplex stability, thus shorter probes can be synthesized.
  • LNA bases render the probe greater duplex stability than the use of single MGB (minor groove binders) at the 3' end. It is an excellent substitute for TaqMan MGB modifications.
  • All combinations of modifications, fluorescent dyes, and backbone modifications can be performed.

SNP Genotyping, Allelic Discrimination

  • LNA substituted bases impart greater specificity with higher Tm.
  • All types of fluorescent dyes and backbone modifications can be performed.
  • 5-Me-dC behaves similar to LNA bases in imparting duplex stability.
  • The high-affinity binding of BNA oligomers has led to develop application for the detection of genetic mutation and mismatch analysis that can use its unique hybridization properties.
  • BNA  has better discrimination power than PNA in real-time PCR application

Hybridization Probes and PCR Amplification Primers

  • LNA substituted bases impart greater specificity with higher Tm. Substitute 4-6 DNA bases with LNA bases.
  • 5-Me-dC behaves similar to LNA bases in imparting duplex stability.
  • BNA enhanced PCR amplification of VNTR locus D1S80 using peptide nucleic acid (BNA).

Bio-Synthesis.Inc, has been producing synthetic oligonucleotides for over 25 years; not only DNA, but RNA, other modified oligonucleotides, and provide cross-linking of various types of biomolecule using our optimized bioconjugation strategies, which have a number of applications on gene expression inhibition and related anti-sense studies. Also synthetic peptides and peptide antibodies for a number of biological relevant applications, in the areas of proteomics, epigenetics, immune regulation, post translational modifications, antisense, gene expression control, RNA interference and more. For more product information, visit: www.biosyn.com.

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