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SPLIT DEOXYRIBOZYME PROBE FOR EFFICIENT DETECTION OF HIGHLY STRUCTURED RNA TARGETS
Title
SPLIT DEOXYRIBOZYME PROBE FOR EFFICIENT DETECTION OF HIGHLY STRUCTURED RNA TARGETS.
Creator
Solarez, Sheila Raquel, Gerasimova, Yulia, De Bekker, Charissa, University of Central Florida
Abstract / Description
Transfer RNAs (tRNAs) are known for their role as adaptors during translation of the genetic information and as regulators for gene expression; uncharged tRNAs regulate global gene expression in response to changes in amino acid pools in the cell. Aminoacylated tRNAs play a role in non-ribosomal peptide bond formation, post-translational protein labeling, modification of phospholipids in the cell membrane, and antibiotic biosynthesis.[1] tRNAs have a highly stable structure that can present a...
Show moreTransfer RNAs (tRNAs) are known for their role as adaptors during translation of the genetic information and as regulators for gene expression; uncharged tRNAs regulate global gene expression in response to changes in amino acid pools in the cell. Aminoacylated tRNAs play a role in non-ribosomal peptide bond formation, post-translational protein labeling, modification of phospholipids in the cell membrane, and antibiotic biosynthesis.[1] tRNAs have a highly stable structure that can present a challenge for their detection using conventional techniques.[2] To enable signal amplification and lower detection limits, a split probe - split deoxyribozyme (sDz or BiDz) probe, which uses a double-labeled fluorogenic substrate as a reporter - has been introduced. In this project we developed an assay based on sDz probe to detect yeast tRNA[Phe] as a proof-of-principle highly structured target. An sDz probe was designed specific to tRNA[phe] that could efficiently unwind stable secondary and tertiary structure of the target RNA thereby providing an efficient tool for tRNA detection.[3] The efficiency of the developed sDz probe was compared with a currently used state-of-the-art hybridization probe - molecular beacon probe. The results obtained in the project further demonstrate the power of sDz probes for the detection of highly structured RNA analytes. The split probes show signal amplification capabilities in detection of structured analytes, which will benefit diagnostics, fundamental molecular biology research and therapeutic fields.
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Date Issued
2018
Identifier
CFH2000311, ucf:45728
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFH2000311
Optimization of Molecular Beacon-Based Multicomponent Probes for Analysis of Nucleic Acids
Title
Optimization of Molecular Beacon-Based Multicomponent Probes for Analysis of Nucleic Acids.
Creator
Stancescu, Maria, Kolpashchikov, Dmitry, Clausen, Christian, Koculi, Eda, Balaeff, Alexander, Shuler, Michael, University of Central Florida
Abstract / Description
Detection of single nucleotide substitutions (SNS) in DNA and RNA has a growing importance in biology and medicine. One traditional approach for recognition of SNS takes advantage of hybridization probes that bind target nucleic acids followed by measuring ?Tm, the difference in melting temperatures of matched and mismatched hybrids. The approach enables SNS differentiation at elevated temperatures (usually 40-65oC) often only in a narrow range of (
Show moreDetection of single nucleotide substitutions (SNS) in DNA and RNA has a growing importance in biology and medicine. One traditional approach for recognition of SNS takes advantage of hybridization probes that bind target nucleic acids followed by measuring ?Tm, the difference in melting temperatures of matched and mismatched hybrids. The approach enables SNS differentiation at elevated temperatures (usually 40-65oC) often only in a narrow range of (<)10oC and requires high-resolution melting devices. Here we demonstrate that a specially designed DNA probe (X sensor) can broaden ?Tm from ~10oC to ~16oC and distinguish SNS in the interval of ~5-40oC. Therefore, there is no need for heating or measuring Tm for accurate SNS differentiation. Our data indicate that this wide differentiation range is in part due to the non-equilibrium hybridization conditions. Further we explored the idea that it is possible to improve the performance of an X sensor operable in close to equilibrium conditions by shifting its operability to non-equilibrium conditions. One way to achieve this is to introduce as many as possible structured ligands in analyte's dissociated state. Here we show that by introducing the maximum possible conformational constraints in X probe it is possible to shift its operation to non-equilibrium conditions and to improve its selectivity at temperatures (<)15oC. Thus, this work points towards a new strategy for the design of highly selective hybridization sensors which operate in non-equilibrium conditions at close to room temperature. The X sensors could be utilized in qPCR, microarrays, as well as RNA analysis in living cells and for ambient temperature point-of-care diagnostics. In the last part of this work, X sensors were used in real time detection of PCR products. The sensors were optimized to operate in PCR buffer with optimal Mg2+ concentration. They were able to detect the target amplicon together with nonspecific products. The results presented here suggest that X sensors might be adopted for real time PCR format.
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Date Issued
2015
Identifier
CFE0006009, ucf:51006
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006009
A TRIPARTILE BIOSENSOR FOR REAL-TIME SNPS DETECTION IN DNA HAIRPIN MOTIF
Title
A TRIPARTILE BIOSENSOR FOR REAL-TIME SNPS DETECTION IN DNA HAIRPIN MOTIF.
Creator
Nguyen, Camha, Kolpashchikov, Dmitry, University of Central Florida
Abstract / Description
The hybridization between two complementary strands of nucleic acid is the basis for a number of applications in DNA and RNA analysis, including in vivo RNA monitoring, microarrays, SNPs detection, and so on. The short oligonucleotide probes form Watson/Crick base pairs (A-T and G-C) with the analyzed nucleic acid. Molecular beacon (MB) probe is one of the most advantageous tools for nucleic acid analysis in real-time. A traditional MB probe consists of a DNA strand folded in hairpin motif...
Show moreThe hybridization between two complementary strands of nucleic acid is the basis for a number of applications in DNA and RNA analysis, including in vivo RNA monitoring, microarrays, SNPs detection, and so on. The short oligonucleotide probes form Watson/Crick base pairs (A-T and G-C) with the analyzed nucleic acid. Molecular beacon (MB) probe is one of the most advantageous tools for nucleic acid analysis in real-time. A traditional MB probe consists of a DNA strand folded in hairpin motif with a fluorophore attached to the 5'end and a quencher attached to the 3' end. The loop segment is complementary to the analytes. Upon hybridization to a complementary single-stranded nucleic acid, MB probe switches to the elongated conformation, which separates the fluorophore from the quencher, resulting in high fluorescence signal. However, DNA or RNA folded in hairpin motifs are difficult to analyze by a conventional MB probes. Inefficient formation of the duplex between the secondary analyte and the MB probe results in low or undetectable fluorescent signal. In this project, we developed a tripartite probe consisting of one MB probe and two adaptor strands to genotype single nucleotide polymorphism (SNPs) in DNA hairpin motifs in real-time fluorescent assays. Each adaptor strand contains a fragment complementary to the analyte and a fragment complementary to an MB probe. One adaptor strand hybridizes to the analyte and unwinds its secondary structure, and the other strand forms stable complex only with the fully complementary analyte sequence. The tri-component probe promises to simplify nucleic acid analysis at ambient temperatures in such application as in vivo RNA monitoring and isothermal detection of specific DNA/RNA targets.
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Date Issued
2011
Identifier
CFH0003768, ucf:44722
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFH0003768