Synthesis and investigation of deoxyribonucleic acid/locked nucleic acid chimeric molecular beacons.
To take full advantage of locked nucleic acid (LNA) based molecular beacons (LNA-MBs) for a variety of applications including analysis of complex samples and intracellular monitoring, we have systematically synthesized a series of DNA/LNA chimeric MBs and studied the effect of DNA/LNA ratio in MBs on their thermodynamics, hybridization kinetics, protein binding affinity and enzymatic resistance. It was found that the LNA bases in a MB stem sequence had a significant effect on the stability of the hair-pin structure. The hybridization rates of LNA-MBs were significantly improved by lowering the DNA/LNA ratio in the probe, and most significantly, by having a shared-stem design for the LNA-MB to prevent sticky-end pairing. It was found that only MB sequences with DNA/LNA alternating bases or all LNA bases were able to resist nonspecific protein binding and DNase I digestion. Additional results showed that a sequence consisting of a DNA stretch less than three bases between LNA bases was able to block RNase H function. This study suggested that a shared-stem MB with a 4 base-pair stem and alternating DNA/LNA bases is desirable for intracellular applications as it ensures reasonable hybridization rates, reduces protein binding and resists nuclease degradation for both target and probes. These findings have implications on the design of LNA molecular probes for intracellular monitoring application, disease diagnosis and basic biological studies.
Yang
Chaoyong James
CJ
Center for Research at the Bio/nano Interface, Department of Chemistry and Shands Cancer Center, UF Genetics Institute and McKnight Brain Institute, University of Florida, Gainesville, FL 32611-7200, USA.
Wang
Lin
L
Wu
Yanrong
Y
Kim
Youngmi
Y
Medley
Colin D
CD
Lin
Hui
H
Tan
Weihong
W
D015342:DNA Probes / Q000737:chemistry; D018076:DNA, Complementary / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry*; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense / Q000737:chemistry* / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism; D013816:Thermodynamics
D015342:DNA Probes; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; C477371:locked nucleic acid; D003850:Deoxyribonuclease I; D016914:Ribonuclease H