PubMed Health⌕ Search

Biomedical subjects

Yingfu Li

Publications and source records attributed to Yingfu Li.

At least 19 recordsLinked to original sources
PubMed2006

Efficient signaling platforms built from a small catalytic DNA and doubly labeled fluorogenic substrates.

RNA-cleaving deoxyribozyme 8-17 has been increasingly used in nanotechnology and biosensing applications. Conventional methods to equip 8-17 with fluorescent signaling property usually involve covalent attachment of two dyes at nucleotide positions that are far away from the catalytic core, such that the bulky dye structures would not affect the deoxyribozyme activity. However, the maximum fluorescent enhancement associated with these 8-17 constructs is typically < or =10-fold, due to a high fluorescent background. To find an optimal balance between signal enhancement and signaling speed, we have conducted a comprehensive study on the effects of the nature of dyes (Alexa Fluor 488, 546 and 647; QSY 9 and 21) as well as their attaching positions along the substrate strand on the catalytic and signaling performance of 8-17. Our results have indicated that 8-17 is able to cleave almost every modified substrate, including those that have chromophores only 1 nt away from the cleavage site. Most importantly, almost all of these substrates are able to generate 15- to 85-fold signal enhancement within 10 min. We have also provided guidelines for selecting substrates that could offer the best signal enhancement, the fastest signaling speed, or the best balance between signal enhancement and signaling speed.

Catalysis↗
PubMed2006

Long-period grating refractive index sensor with a modified cladding structure for large operational range and high sensitivity.

What is believed to be a novel long-period grating (LPG) refractive index sensor with a modified cladding structure is proposed and studied. In the proposed structure, the cladding of the fiber has a two-layer structure, i.e., a cladding layer of low refractive index with a reduced radius and an overlay of high refractive index. The sensitivity of the structure-modified LPG sensor to the ambient refractive index change as a function of the cladding layer and overlay parameters is investigated by way of modeling. It is found that an increase of the ambient refractive index causes a field redistribution of the cladding mode into the overlay when the parameters of the overlay are properly selected. It is shown that by reducing the radius of the cladding layer, the operational range of the LPG refractive index sensor can be as large as 0.195 (from 1.244 to 1.440) with a minimum sensitivity of 660 nm/refractive index, which represents a 31% increase of operational range in comparison with the operational range obtained from the reported structure. The design guidelines for achieving this large operation range and high sensitivity are explained by investigating the dependence of the cladding modes on the radius of the cladding layer.

Journal Article↗
PubMed2006

Phosphoester-transfer mechanism of an RNA-cleaving acidic deoxyribozyme revealed by radioactivity tracking and enzymatic digestion.

A convenient method involving 32P-labeling of an RNA substrate at the cleavage site and subsequent enzymatic digestion of cleavage products via phosphatases reveals that pH4DZ1--an RNA-cleaving deoxyribozyme with optimal activity at pH 4--forms a 5'-cleavage fragment with 2',3'-cyclic phosphate group and a 3'-cleavage fragment with 5'-OH group.

DNA, Catalytic↗
PubMed2006

Characterization of long RNA-cleaving deoxyribozymes with short catalytic cores: the effect of excess sequence elements on the outcome of in vitro selection.

We previously conducted an in vitro selection experiment for RNA-cleaving deoxyribozymes, using a combinatorial DNA library containing 80 random nucleotides. Ultimately, 110 different sequence classes were isolated, but the vast majority contained a short14-15 nt catalytic DNA motif commonly known as 8-17. Herein, we report extensive truncation experiments conducted on multiple sequence classes to confirm the suspected catalytic role played by 8-17 and to determine the effect of excess sequence elements on the activity of this motif and the outcome of selection. Although we observed beneficial, detrimental and neutral consequences for activity, the magnitude of the effect rarely exceeded 2-fold. These deoxyribozymes appear to have survived increasing selection pressure despite the presence of additional sequence elements, rather than because of them. A new deoxyribozyme with comparable activity, called G15-30, was approximately 2.5-fold larger and experienced a approximately 4-fold greater inhibitory effect from excess sequence elements than the average 8-17 motif. Our results suggest that 8-17 may be less susceptible to the potential inhibitory effects of excess arbitrary sequence than larger motifs, which represents a previously unappreciated selective advantage that may contribute to its widespread recurrence.

Base Sequence↗
PubMed2006

Nucleic acid aptamers and enzymes as sensors.

The function of nucleic acids has been an endless source of discovery and invention that has drastically enhanced our appreciation of DNA and RNA as multifaceted polymers. It is now widely known that nucleic acids can act as enzymes (deoxyribozymes and ribozymes) and as receptors (aptamers), and that these functional nucleic acids (FNAs) can either be found in nature or isolated from pools of random nucleic acids. The availability of many natural and artificial FNAs has opened a new horizon for the development of 'smart' molecules for a variety of chemical and biological applications. This review provides a snapshot of recent progress in the application of FNAs as novel sensors for biomolecular detection, drug discovery and nanotechnology.

Aptamers, Nucleotide↗
PubMed2006

Revitalization of six abandoned catalytic DNA species reveals a common three-way junction framework and diverse catalytic cores.

A library containing as many as 10(16) nucleic acid candidates is typically used to isolate artificial ribozymes and deoxyribozymes (DNAzymes) in an in vitro selection experiment, with only a handful of sequences surviving many rounds of stringent selection steps. These winning species are generally the focus of interest whereas the less competitive contenders are usually not examined. Nevertheless, molecular species abandoned during the selection process might still represent a rich pool of catalytic motifs that are useful for the examination of DNA's inherent catalytic ability, and for the design of molecular tools for practical applications. Here we report a study of six RNA-cleaving, fluorescence-signaling deoxyribozymes that appeared in the early generations of a previous in vitro selection experiment, using the combined approaches of reselection, rational structural analysis, and reaction condition optimization. All six deoxyribozymes were found to use a three-way junction as a common structural framework for catalysis. However, disparities observed in the conserved nucleotide allocations, methylation interference patterns and metal-ion selectivities, pointed to distinct catalytic cores. The rate constants of the optimized deoxyribozymes fell in the range of approximately 0.2 to 1.6 min(-1), which are comparable to those of similar ribozymes. Our findings indicate that deoxyribozymes eliminated by harsh selection criteria are structurally simple molecules that can be tailored into efficient catalysts.

Base Sequence↗
PubMed2006

Quenching of fluorophore-labeled DNA oligonucleotides by divalent metal ions: implications for selection, design, and applications of signaling aptamers and signaling deoxyribozymes.

Recent years have seen a dramatic increase in the use of fluorescence-signaling DNA aptamers and deoxyribozymes as novel biosensing moieties. Many of these functional single-stranded DNA molecules are either engineered to function in the presence of divalent metal ion cofactors or designed as sensors for specific divalent metal ions. However, many divalent metal ions are potent fluorescence quenchers. In this study, we first set out to examine the factors that contribute to quenching of DNA-bound fluorophores by commonly used divalent metal ions, with the goal of establishing general principles that can guide future exploitation of fluorescence-signaling DNA aptamers and deoxyribozymes as biosensing probes. We then extended these studies to examine the effect of specific metals on the signaling performance of both a structure-switching signaling DNA aptamer and an RNA-cleaving and fluorescence-signaling deoxyribozyme. These studies showed extensive quenching was obtained when using divalent transition metal ions owing to direct DNA-metal ion interactions, leading to combined static and dynamic quenching. The extent of quenching was dependent on the type of metal ion and the concentration of supporting monovalent cations in the buffer, with quenching increasing with the number of unpaired electrons in the metal ion and decreasing with the concentration of monovalent ions. The extent of quenching was independent of the fluorophore, indicating that quenching cannot be alleviated simply by changing the nature of the fluorescent probe. Our results also show that the DNA sequence and the local secondary structure in the region of the fluorescent tag can dramatically influence the degree of quenching by divalent transition metal ions. In particular, the extent of quenching is predominantly determined by the fluorophore location with respect to guanine-rich and duplex regions within the strand sequence. Examination of the effect of both the type and concentration of metal ions on the performance of a fluorescence-signaling aptamer and a signaling deoxyribozyme confirms that judicious choice of divalent transition metal ions is important in maximizing signals obtained from such systems.

Aptamers, Nucleotide↗
PubMed2006

Characterization of a catalytically efficient acidic RNA-cleaving deoxyribozyme.

We previously demonstrated--through the isolation of RNA-cleaving deoxyribozymes by in vitro selection that are catalytically active in highly acidic solutions--that DNA, despite its chemical simplicity, could perform catalysis under challenging chemical conditions [Liu,Z., Mei,S.H., Brennan,J.D. and Li,Y. (2003) J. Am. Chem. Soc. 125, 7539-7545]. One remarkable DNA molecule therefrom is pH4DZ1, a self-cleaving deoxyribozyme that exhibits a k(obs) of approximately 1 min(-1) at pH 3.8. In this study, we carried out a series of experiments to examine the sequence and catalytic properties of this acidic deoxyribozyme. Extensive nucleotide truncation experiments indicated that pH4DZ1 was a considerably large deoxyribozyme, requiring approximately 80 out of the original 123 nt for the optimal catalytic activity. A reselection experiment identified ten absolutely conserved nucleotides that are distributed in three catalytically crucial sequence elements. In addition, a trans deoxyribozyme was successfully designed. Comparison of the observed rate constant of pH4DZ1 with experimentally determined rate constant for the uncatalyzed reaction revealed that pH4DZ1 achieved a rate enhancement of approximately 10(6)-fold. This study provides valuable information about this low-pH-functional deoxyribozyme and paves way for further structural and mechanistic characterization of this unique catalytic DNA.

Base Sequence↗
PubMed2006

Evolution of high-branching deoxyribozymes from a catalytic DNA with a three-way junction.

Here, we report the evolution of two star-shaped (five-way junction) deoxyribozymes from a catalytic DNA containing a three-way junction scaffold. The transition was shown to be a switch rather than a gradual progression. The star-shaped motifs, surprisingly, only took five selection cycles to be detected, and another four to dominate the evolving population. Chemical probing experiments indicated that the two deoxyribozymes belong to the same family despite noticeable variations in both the primary sequence and the secondary structure. Our findings not only describe the evolution of high-branching nucleic acid structures from a low-branching catalytic module, but they also illustrate the idea of deriving a rare structural motif by sampling the sequence variants of a given functional nucleic acid.

Base Sequence↗
PubMed2005

Aptamers with fluorescence-signaling properties.

Aptamers are single-stranded DNA or RNA molecules with ligand-binding capabilities. Signaling aptamers refer to aptamers or modified aptamers with recordable signal generation ability. Fluorescence-signaling aptamers, in particular, are valuable molecular tools that can be used to establish important techniques or assays for the interrogation of identities and concentrations of proteins and metabolites. Since standard DNA and RNA aptamers themselves are not inherently fluorescent, modification methods are required for rationally converting non-fluorescent aptamers into fluorescent reporters or for selecting fluorescent aptamers directly from random-sequence DNA libraries by in vitro selection. This article will provide a brief review of various signaling aptamer design strategies as well as a detailed description of methods that can be used to generate, by both rational design and in vitro selection, a special class of signaling aptamers dubbed "structure-switching signaling aptamers." This class of signaling aptamers are designed to function by switching structures from a pre-formed, lowly fluorescent duplex assembly to a ligand-aptamer complex having a higher level of fluorescence.

Adenosine Triphosphate↗
PubMed2005

Characterizing the secondary structure and identifying functionally essential nucleotides of pH6DZ1, a fluorescence-signaling and RNA-cleaving deoxyribozyme.

pH6DZ1 is a synthetic deoxyribozyme that is able to couple catalysis with fluorescence signal generation. This deoxyribozyme has the ability to cleave itself at a lone ribonucleotide that is present between a pair of deoxyribothymidines, one modified with a fluorophore (fluorescein) and the other with a quencher (DABCYL). Herein, we report on the sequence truncation and secondary structure characterization of pH6DZ1 as well as the identification of functionally important nucleotides within this deoxyribozyme. Our data indicate that pH6DZ1 has a four-way, junction-like secondary structure comprised of four short duplexes, three hairpin loops, and three interhelical unpaired elements. Ten nucleotides, all located in two separate single-stranded regions, were identified as functionally indispensable nucleotides (complete loss of the catalytic function was obtained upon mutation). Nine nucleotides, most of which are also distributed in three single-stranded DNA elements, were identified as functionally vital nucleotides (at least a 1000-fold activity reduction was obtained upon mutation). Our study has shown that pH6DZ1 has a secondary structure that is more complex than those reported for other RNA-cleaving deoxyribozymes. The identification of functionally important nucleotides lays the foundation for future mechanistic studies on this DNAzyme. The elucidation of the secondary structure of pH6DZ1 should facilitate the future exploration of this unique DNAzyme for the development of DNAzyme-based biosensors.

Base Sequence↗
PubMed2005

Entrapment of fluorescent signaling DNA aptamers in sol-gel-derived silica.

We report on the first successful immobilization of a DNA aptamer, in particular, a fluorescence-signaling DNA aptamer, within a sol-gel-derived matrix. The specific aptamer examined in this study undergoes a structural switch in the presence of adenosine triphosphate (ATP) to release a dabcyl-labeled nucleotide strand (QDNA), which in turn relieves the quenching of a fluorescein label that is also present in the aptamer structure. It was demonstrated that aptamers containing a complementary QDNA strand along with either a short complimentary strand bearing fluorescein (tripartite structure) or a directly bound fluorescein moiety (bipartite structure) remained intact upon entrapment within biocompatible sol-gel derived materials and retained binding activity, structure-switching capabilities, and fluorescence signal generation that was selective and sensitive to ATP concentration. Studies were undertaken to evaluate the properties of the immobilized aptamers that were either in their native state or bound to streptavidin using a terminal biotin group on the aptamer, including response time, accessibility, and leaching. Furthermore, signaling abilities were optimized through evaluation of different QDNA constructs. These studies indicated that the aptamers remained in a state that was similar to solution, with moderate leaching, only minor decreases in accessibility to ATP, and an expected reduction in response time due to diffusional barriers to mass transport of the analyte through the silica matrix. Entrapment of the aptamer also resulted in protection of the DNA against degradation from nucleases, improving the potential for use of the aptamer for in vivo sensing. This work demonstrates that sol-gel-derived materials can be used to successfully immobilize and protect DNA-based biorecognition elements and, in particular, DNA aptamers, opening new possibilities for the development of DNA aptamer-based devices, such as affinity columns, microarrays, and fiber-optic sensors.

Adenosine Triphosphate↗