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Detection of endogenous k-RAS mRNA in living cells at a single base resolution by a PNA molecular beacon.
MedLine Citation:
PMID:  22289057     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
Detection of mRNA alterations is a promising approach for identifying biomarkers as means of differentiating benign from malignant lesions. By choosing the K-ras oncogene as a target gene, two types of molecular beacons (MBs) based on either phosphothioated DNA (PS-DNA-MB) or peptide nucleic acid (TO-PNA-MB, where TO = thiazole orange) were synthesized and compared in vitro and in-vivo. Their specificity was examined in wild-type K-Ras (HT29) or codon 12 point mutations (Panc-1, SW480) cells. Incubation of both beacons with total RNA extracted from the Panc-1 cell line (fully complement sequence) showed a fluorescent signal for both beacons. Major differences were observed, however, for single mismatch mRNA transcripts in cell lines HT29 and SW480. PS-DNA-MB weakly discriminated such single mismatches in comparison to TO-PNA-MB that was profoundly more sensitive. Cell transfection of TO-PNA-MB with the aid of PEI resulted in fluorescence in cells expressing the fully complementary RNA transcript (Panc-1) but undetectable fluorescence in cells expressing the k-RAS mRNA that has a single mismatch to the designed TO-PNA-MB (HT29). A weaker fluorescent signal was also detected in SW480 cells; however, these cells express approximately one fifth of the target mRNA of the designed TO-PNA-MB. In contrast, PS-DNA-MB showed no fluorescence in all cell lines tested post PEI transfection. Based on the fast hybridization kinetics and on the single mismatch discrimination found for TO-PNA-MB we believe that such molecular beacons are promising for in-vivo real-time imaging of endogenous mRNA with single nucleotide polymorphism (SNP) resolution.
Authors:
Yossi Kam; Abraham Rubinstein; Aviram Nissan; David Halle; Eylon Yavin
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-1-30
Journal Detail:
Title:  Molecular pharmaceutics     Volume:  -     ISSN:  1543-8392     ISO Abbreviation:  -     Publication Date:  2012 Jan 
Date Detail:
Created Date:  2012-1-31     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101197791     Medline TA:  Mol Pharm     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
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