locked nucleic acid - novel dna anolog mec meeting hanyang university proteome research lab 2002....

Locked Nucleic Acid- Novel DNA Anolog

MEC Meeting

Hanyang University Proteome Research Lab

2002. 12. 13

Park, Ji-Yoon

Proteome Research Lab, http://gene.hanyang.ac.kr Proteome Research Lab, http://gene.hanyang.ac.kr

Nucleic Acid Analogs

Proteome Research Lab, http://gene.hanyang.ac.kr Proteome Research Lab, http://gene.hanyang.ac.kr

Comparison of LNA and PNA

Proteome Research Lab, http://gene.hanyang.ac.kr Proteome Research Lab, http://gene.hanyang.ac.kr

Locked DNA Analog(LNA)Locked DNA Analog(LNA)

Novel chemical DNA Novel chemical DNA Anolog Anolog

Bicyclic nucleic acid Bicyclic nucleic acid A ribonucleoside is linked A ribonucleoside is linked

between the 2’ oxygen between the 2’ oxygen and the 4’ carbon atoms and the 4’ carbon atoms with a methylene unit with a methylene unit

Proteome Research Lab, http://gene.hanyang.ac.kr Proteome Research Lab, http://gene.hanyang.ac.kr

General PropertiesGeneral Properties

Base pairs with DNA and RNABase pairs with DNA and RNA

Obey standard WC base pairing ruleObey standard WC base pairing rule

Exceptionally high thermal stabilityExceptionally high thermal stability

Improved discriminationImproved discrimination

Compatible with most enzymesCompatible with most enzymes

Predictable melting behaviourPredictable melting behaviour

Synthesis by standard phosphoramidite chemistry using Synthesis by standard phosphoramidite chemistry using DNA-synthesizersDNA-synthesizers

mixed with DNA, RNA and other nucleic acid analogsmixed with DNA, RNA and other nucleic acid analogs

synthesized with e.g. aminolinkers, biotin, Cy dyes etcsynthesized with e.g. aminolinkers, biotin, Cy dyes etc

water soluble(gel electrophoresis, ethanol precipitation)

Proteome Research Lab, http://gene.hanyang.ac.kr Proteome Research Lab, http://gene.hanyang.ac.kr

Experimental Parameters or Application

Proteome Research Lab, http://gene.hanyang.ac.kr Proteome Research Lab, http://gene.hanyang.ac.kr

Melting Temperature( Tm )Melting Temperature( Tm )

Proteome Research Lab, http://gene.hanyang.ac.kr Proteome Research Lab, http://gene.hanyang.ac.kr

Nearest Neighbor ThermodynamicsNearest Neighbor Thermodynamics

Proteome Research Lab, http://gene.hanyang.ac.kr Proteome Research Lab, http://gene.hanyang.ac.kr

Fluorescence Microscope for generating Melting Curve

Fluorescence measurement in real-time PCR instrument Fluorescence measurement in real-time PCR instrument

Decrease in fluorescence intensity from specific binding to ds DNADecrease in fluorescence intensity from specific binding to ds DNA

Proteome Research Lab, http://gene.hanyang.ac.kr Proteome Research Lab, http://gene.hanyang.ac.kr

How LNA Substitution Increase ∆Tm

Proteome Research Lab, http://gene.hanyang.ac.kr Proteome Research Lab, http://gene.hanyang.ac.kr

Comparison of LNA and DNA Stability

Proteome Research Lab, http://gene.hanyang.ac.kr Proteome Research Lab, http://gene.hanyang.ac.kr

Effect of Single LNA Substitution

Proteome Research Lab, http://gene.hanyang.ac.kr Proteome Research Lab, http://gene.hanyang.ac.kr

www.LNA-Tm.com

Proteome Research Lab, http://gene.hanyang.ac.kr Proteome Research Lab, http://gene.hanyang.ac.kr

Sensitivity Test LNA Capture Probes(50mer) vs DNA Capture Probe(50mer)

< Hybridization condition>< Hybridization condition> A) 65 A) 65°C, in 3x SSC °C, in 3x SSC

B) B) Hybridization: 70°C, in 3x SSCHybridization: 70°C, in 3x SSC

Proteome Research Lab, http://gene.hanyang.ac.kr Proteome Research Lab, http://gene.hanyang.ac.kr

Specificity Test LNA Capture Probes(40mer) vs DNA Capture Probe(40mer)

• Hybridization condition: 65°C, in 3x SSCHybridization condition: 65°C, in 3x SSC - Perfect match: 1-7- Perfect match: 1-7 - Mismatch: 2-8, 3-9, 4-10, 5-11, 6-12 - Mismatch: 2-8, 3-9, 4-10, 5-11, 6-12

Proteome Research Lab, http://gene.hanyang.ac.kr Proteome Research Lab, http://gene.hanyang.ac.kr

Microfluidic Chip in Action