An Efficient RNA-Cleaving DNA Enzyme that Synchronizes Catalysis with Fluorescence Signaling

Mei, Shirley H. J.; Liu, Zhongjie; Brennan, John D.; Li, Yingfu

doi:10.1021/ja0281232

Cited by 194 publications

(203 citation statements)

References 32 publications

(19 reference statements)

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“…However, from the sixth round, the C DNA i -L DNA ii hybridization time, T1, and the DNA ligation time, T2, were shortened as follows: round 6, T1 ¼ 1,200 min and T2 ¼ 60 min; round 7, T1 ¼ 300 min and T2 ¼ 60 min; round 8, T1 ¼ 60 min and T2 ¼ 60 min; round 9, T1 ¼ 10 min and T2 ¼ 60 min; round 10, T1 ¼ 10 min and T2 ¼ 20 min; round 11, T1 ¼ 2 min and T2 ¼ 20 min; round 12, T1 ¼ 30 s and T2 ¼ 20 min; rounds 13-15, T1 ¼ 30 s and T2 ¼ 10 min. DNA sequences from the 15th round were amplified by PCR and cloned by the TA cloning method as previously described 32 .…”

Section: Methodsmentioning

confidence: 99%

Engineering interlocking DNA rings with weak physical interactions

Shen

Tram

et al. 2014

Nat Commun

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Catenanes are intriguing molecular assemblies for engineering unique molecular devices. The resident rings of a catenane are expected to execute unhindered rotation around each other, and to do so, they must have weak physical interactions with each other. Due to sequence programmability, DNA has become a popular material for nanoscale object engineering. However, current DNA catenanes, particularly in the single-stranded (ss) form, are synthesized through the formation of a linking duplex, which makes them less ideal as mobile elements for molecular machines. Herein we adopt a random library approach to engineer ssDNA [2] catenanes (two interlocked DNA rings) without a linking duplex. Results from DNA hybridization, double-stranded catenane synthesis and rolling circle amplification experiments signify that representative catenanes have weak physical interactions and are capable of operating as independent units. Our findings lay the foundation for exploring free-functioning interlocked DNA rings for the design of elaborate nanoscale machines based on DNA.

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Section: Methodsmentioning

confidence: 99%

Engineering interlocking DNA rings with weak physical interactions

Shen

Tram

et al. 2014

Nat Commun

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“…1) and investigated the use of RFDs as analytical tools. [17][18][19][20][21][22][23][24][25][26][27][28][29] RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence.…”

Section: -8mentioning

confidence: 99%

“…FS1 was obtained from Keck Oligo Synthesis Facilities at Yale University, deprotected and purified by gel electrophoresis following a previously established protocol. [17][18][19][20][21][22][23][24] EC1, SS1 and LT1 were purchased from Integrated DNA Technologies and purified by gel electrophoresis. vortexing and place the tubes in -20 °C freezer for 30 min.…”

Section: Construction Of Rfd-ec1 and Rfss1 By Template Mediated Enzymmentioning

confidence: 99%

Detection of Bacteria Using Fluorogenic DNAzymes

Aguirre

Ali

Kanda

et al. 2012

JoVE

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Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-toresults (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR-and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment. 2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection. 5DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions. [6][7][8] These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 10 16 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment). 9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications. 6-8Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools. [17][18][19][20][21][22][23][24][25][26][27][28][29] RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.More recently, we developed a method of isolating RFDs for bacterial detection. 5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifyi...

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“…DNAzymes have also been developed that combine RNA cleavage with fluorescence signaling Mei et al, 2003). This arrangement allows the cleavage reaction to be monitored in real time.…”

Section: Other Rna-cleaving Deoxyribozymesmentioning

confidence: 99%