Search PubMed⌕ Search

Biomedical subjects

Yingfu Li

Publications and source records attributed to Yingfu Li.

At least 37 records · Page 2Linked to original sources
PubMed2005

Diverse evolutionary trajectories characterize a community of RNA-cleaving deoxyribozymes: a case study into the population dynamics of in vitro selection.

Two parallel in vitro selections (denoted Selection A and Selection B) were conducted under different selection-pressure regimes, yielding a diverse community of RNA-cleaving deoxyribozymes. In Selection A, the reaction time was reduced four times (from 5 h to 5 s) over the course of 24 generations, while in Selection B the reaction time was maintained at 5 h for 30 rounds of selective amplification. Sequence alignment was conducted on more than 800 clones assembled from 18 generations that span both selections. Many prominent catalytic sequence classes, including some that extend across both selections, were identified and used to construct fitness landscapes depicting their rise and fall over time. The landscapes from both selections exhibit similar global trends despite differences in population dynamics. Some deoxyribozymes were predominant in the early rounds of selection but gave way to other species that dominated in the middle rounds. Ultimately, these middle classes disappeared from the landscape in favor of new and presumably more fit deoxyribozyme sequence classes. The shape of these landscapes alludes to the presence of many latent deoxyribozymes in the initial library, which can only be accessed by changes in the selection pressure and/or by adaptive mutations. Basic computer simulations provide theoretical corroboration of the experimentally observed pattern of staggered sequence-class transitions across the fitness landscapes. These simulations model the influence of one or more contributing factors, including catalytic rate, folding efficiency, PCR amplification efficiency, and random mutagenesis. This is the first study which thoroughly documents the topography of a deoxyribozyme fitness landscape over many generations of in vitro selection.

Base Sequence↗
PubMed2005

Benzimidazoles as non-peptide luteinizing hormone-releasing hormone (LHRH) antagonists. Part 3: Discovery of 1-(1H-benzimidazol-5-yl)-3-tert-butylurea derivatives.

1-(1H-Benzimidazol-5-yl)-3-tert-butylurea derivatives have been identified as a novel class of non-peptide luteinizing hormone-releasing hormone (LHRH) antagonists. Herein, we disclose the synthesis and structure-activity relationships (SAR) of this class resulting in the identification of compound 12c, with dual functional activity on human and rat receptors (rat LHRH: IC50=120 nM; human LHRH: IC50=18 nM). These SAR studies suggest that 1-(1H-benzimidazol-5-yl)-3-tert-butylurea is a new pharmacophore for small molecule LHRH antagonists.

Animals↗
PubMed2005

Secondary-structure characterization of two proficient kinase deoxyribozymes.

Dk1 and Dk2 are two catalytically proficient, manganese-dependent, guanine-rich deoxyribozymes previously isolated for DNA phosphorylation. In this study, we carried out a series of experiments that aimed to understand the structural properties of Dk1 and Dk2 and compare the structural similarities or differences of these two distinct deoxyribozymes that carry out similar catalytic functions. First, we performed reselections from two partially randomized DNA libraries on the basis of the original Dk1 and Dk2 sequences to isolate catalytically active sequence variants and identify nucleotides that are invariable, well-conserved, or highly mutagenized. Sequence analysis of these variants assisted by secondary-structure predictions led to the identification of possible Watson-Crick base-pairing regions within each deoxyribozyme. Sequence truncation and base-pair partner exchange experiments were conducted to confirm, or rule out, the existence of the predicted secondary-structure elements. Finally, methylation interference experiments were applied to identify nucleotides that are potentially important for the tertiary structure folding of the deoxyribozymes. Our data suggest that Dk1 and Dk2, despite the differences in their primary sequences and NTP requirements, use an analogous stem-loop element to anchor a structural domain of substantial tertiary interactions to execute their catalytic functions.

Base Pair Mismatch↗
PubMed2005

Benzimidazole derivatives as novel nonpeptide luteinizing hormone-releasing hormone (LHRH) antagonists. Part 2: Benzimidazole-5-sulfonamides.

The 2-cyclopropyl substituted benzimidazole 2 has been used as a starting point for further optimization of an LHRH antagonist series. SAR studies revealed that a tert-butyl urea fragment connected through a simple carbon chain would improve activity. Further modification of the benzylsulfonamide moiety led to the discovery of 23 (IC(50): 4.2 nM).

Benzimidazoles↗
PubMed2005

Tau protein binds single-stranded DNA sequence specifically--the proof obtained in vitro with non-equilibrium capillary electrophoresis of equilibrium mixtures.

Tau is a microtubule-associated protein, which plays an important role in physiology and pathology of neurons. Tau has been recently reported to bind double-stranded DNA (dsDNA) but not to bind single-stranded DNA (ssDNA) [Cell. Mol. Life Sci. 2003, 60, 413-421]. Here, we prove that tau binds not only dsDNA but also ssDNA. This finding was facilitated by using two kinetic capillary electrophoresis methods: (i) non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM); (ii) affinity-mediated NECEEM. Using the new approach, we observed, for the first time, that tau could induce dissociation of strands in dsDNA by binding one of them in a sequence-specific fashion. Moreover, we determined the equilibrium dissociation constants for all tau-DNA complexes studied.

Animals↗
PubMed2005

Characterization of a fluorescence-signaling and RNA-cleaving deoxyribozyme.

DNA enzymes (deoxyribozymes or DNAzymes) are single-stranded DNA molecules with catalytic function. Previously, we isolated several RNA-cleaving deoxyribozymes with fluorescence-signaling properties. These special DNA molecules are capable of cleaving an RNA linkage embedded within a DNA sequence and flanked by a pair of deoxyribothymidines modified with a fluorophore (fluorescein) and a quencher (dabcyl). Here we report on the sequence truncation and secondary structure characterization of one such deoxyribozyme known as pH6DZ1 as well as the identification of functionally important nucleotides within this deoxyribozyme. Our data indicates that pH6DZ1 has a four-way junction-like secondary structure comprising four short duplexes, three hairpin loops, and three inter-helical unpaired elements.

Base Sequence↗
PubMed2005

Benzimidazole-5-sulfonamides as novel nonpeptide luteinizing hormone releasing hormone (LHRH) antagonists: minimization of mechanism-based CYP3A4 inhibition.

Herein we report the development of novel, potent and non-peptide luteinizing hormone releasing hormone (LHRH) antagonists. The optimization towards derivatives free from mechanism-based CYP3A4 inhibition is described. The identification of a main metabolite guided us towards structural modifications of the benzyl moiety, which resulted in significant improvements of the CYP3A4 profile, while maintaining potent LHRH antagonist activity.

Animals↗
PubMed2004

Tracing sequence diversity change of RNA-cleaving deoxyribozymes under increasing selection pressure during in vitro selection.

In vitro selection has been used extensively over the past 10 years to create functionally diverse DNA enzymes. The majority of in vitro selection experiments to date have focused on the outcome rather than the process itself, a process that remains to be fully elucidated. In vitro selection techniques rely on the probability that some DNA molecules in a random-sequence library will fold into an appropriate tertiary structure and catalyze a desired reaction. Thus, sufficient sequence diversity in the DNA pool (and hence more catalytic DNA sequences) is a prerequisite for the successful isolation of efficient deoxyribozymes. The catalytic sequence diversity established by in vitro selection is governed largely by the choice of selection pressures, one of which is the length of the reaction time. The objective of this study was to evaluate the sequence diversity change of a pool of RNA-cleaving deoxyribozymes as a function of the reaction time. Seventeen rounds of in vitro selection were performed, and the reaction time was progressively decreased from 5 h to 5 s. A representative population from each time class was subsequently cloned and sequenced. A decline in sequence diversity was observed with decreasing reaction time, and the relationship appears to be logarithmic. In contrast, a control selection performed with a constant reaction time during each round led to a linear and comparatively very slow decrease in sequence diversity. This study provides the first methodical examination of the change in catalytic sequence diversity that occurs through the course of a deoxyribozyme selection experiment. Moreover, it represents a first step toward fully understanding the intricate pathway that lies between the beginning and end of an in vitro selection experiment.

Base Sequence↗
PubMed2004

Structure-switching signaling aptamers: transducing molecular recognition into fluorescence signaling.

The development of aptamer technology considerably broadens the utility of nucleic acids as molecular recognition elements, because it allows the creation of DNA or RNA molecules for binding a wide variety of analytes (targets) with high affinity and specificity. Several recent studies have focused on developing rational design strategies for transducing aptamer-target recognition events into easily detectable signals, so that aptamers can be widely exploited for detection directed applications. We have devised a generalizable strategy for designing nonfluorescent aptamers that can be turned into fluorescence-signaling reporters. The resultant signaling probes are denoted "structure-switching signaling aptamers" as they report target binding by switching structures from DNA/DNA duplex to DNA/target complex. The duplex is formed between a fluorophore-labeled DNA aptamer and an antisense DNA oligonucleotide modified with a quencher (denoted QDNA). In the absence of the target, the aptamer hybridizes with QDNA, bringing the fluorophore into close proximity of the quencher for efficient fluorescence quenching. When this system is exposed to the target, the aptamer switches its binding partner from QDNA to the target. This structure-switching event is coupled to the generation of a fluorescent signal through the departure of QDNA, permitting the real-time monitoring of the aptamer-target recognition. In this article, we discuss the conceptual framework of the structure-switching approach, the essential features of structure-switching signaling aptamers as well as remaining challenges and possible solutions associated with this new methodology.

Adenosine Triphosphate↗
PubMed2004

Ligating DNA with DNA.

Cloning DNA typically involves the joining of target DNAs with vector constructs by enzymatic ligation. A commonly used enzyme for this reaction is bacteriophage T4 DNA ligase, which requires ATP as the energy source to catalyze the otherwise unfavorable formation of a phosphodiester bond. Using in vitro selection, we have isolated a DNA sequence that catalyzes the ligation of DNA in the absence of protein enzymes. We have used the action of two catalytic DNAs, an ATP-dependent self-adenylating deoxyribozyme (AppDNA) and a self-ligating deoxyribozyme, to create a ligation system that covalently joins oligonucleotides via the formation of a 3',5'-phosphodiester linkage. The two-step process is conducted in separate reaction vessels wherein the products of deoxyribozyme adenylation are purified before their use as substrates for deoxyribozyme ligation. The final ligation step of the deoxyribozyme-catalyzed sequence of reactions mimics the final step of the T4 DNA ligase reaction. The initial rate constant (k(obs)) of the optimized deoxyribozyme ligase was found to be 1 x 10(-)(4) min(-)(1). Under these conditions, the ligase deoxyribozyme promotes DNA ligation at least 10(5)-fold faster than that generated by a simple DNA template. The self-ligating deoxyribozyme has also been reconfigured to generate a trans-acting construct that joins separate DNA oligonucleotides of defined sequence. However, the sequence requirements of the AppDNA and that of the 3' terminus of the deoxyribozyme ligase limit the range of sequences that can be ligated.

Base Sequence↗
PubMed2004

Synthesis and characterization of topologically linked single-stranded DNA rings.

We investigated the synthesis of linked-ring DNAs by two DNA-ligation-based methods. In the first method, two DNA oligonucleotides were associated through a duplex segment of more than a full helical turn. Circularization of the entwined oligonucleotides by T4 DNA ligase resulted in two linked-ring DNAs with a total yield of approximately 40%. In the second method, a DNA oligonucleotide was circularized over a circular DNA template, resulting in the formation of approximately 10% linked-ring product. The circular nature of linked-ring DNAs was verified with exonuclease digestion and the existence of topological linkages was demonstrated by analyzing the electrophoretic mobility pattern of DNA products obtained from the digestion of each linked-ring DNA using specific restriction endonucleases. A linked-ring DNA library in which one of the two rings contained random-sequence nucleotides was also constructed and tested for compatibility with in vitro selection.

Base Pairing↗
PubMed2004

Dinucleotide junction cleavage versatility of 8-17 deoxyribozyme.

We conducted 16 parallel in vitro selection experiments to isolate catalytic DNAs from a common DNA library for the cleavage of all 16 possible dinucleotide junctions of RNA incorporated into a common DNA/RNA chimeric substrate sequence. We discovered hundreds of sequence variations of the 8-17 deoxyribozyme--an RNA-cleaving catalytic DNA motif previously reported--from nearly all 16 final pools. Sequence analyses identified four absolutely conserved nucleotides in 8-17. Five representative 8-17 variants were tested for substrate cleavage in trans, and together they were able to cleave 14 dinucleotide junctions. New 8-17 variants required Mn2+ to support their broad dinucleotide cleavage capabilities. We hypothesize that 8-17 has a tertiary structure composed of an enzymatic core executing catalysis and a structural facilitator providing structural fine tuning when different dinucleotide junctions are given as cleavage sites.

Catalysis↗
PubMed2003

Assemblage of signaling DNA enzymes with intriguing metal-ion specificities and pH dependences.

We report a group of new DNA enzymes that possess a synchronized RNA-cleavage/fluorescence-signaling ability and exhibit wide-ranging metal-ion and pH dependences. These DNA catalysts were derived from a random-sequence DNA pool in a two-stage process: (1) establishment of a catalytic DNA population through repetitive rounds of in vitro selection at pH 4.0, and (2) sequence-diversification and catalytic-activity optimization through five parallel paths of in vitro evolution conducted at pH 3.0, 4.0, 5.0, 6.0, and 7.0, respectively. The deoxyribozymes were evolved to cleave the phosphodiester bond of a single ribonucleotide embedded in DNA and flanked immediately by two deoxyribonucleotides modified with a fluorophore and a quencher, respectively--a setting that synchronizes catalysis with fluorescence signaling. The most dominant catalyst from each pool was examined for metal-ion specificity, catalytic efficiency, pH dependence, and fluorescence-signaling capability. Individual catalysts have different metal-ion requirements and can generate as much as a 12-fold fluorescence enhancement upon RNA cleavage. Most of the DNA enzymes have a pH optimum coinciding with the selection pH and exhibit a rate constant approximating 1 min(-)(1) under optimal reaction conditions. The demonstration of DNA enzymes that are functional under extremely high acidity (such as pH 3 and 4) indicates that DNA has the ability to perform efficient catalysis even under harsh reaction conditions. The isolation of many new signaling DNA enzymes with broad pH optima and metal-ion specificities should facilitate the development of diverse deoxyribozyme-based biosensors.

Base Sequence↗
PubMed2003

Structure-switching signaling aptamers.

Aptamers are single-stranded nucleic acids with defined tertiary structures for selective binding to target molecules. Aptamers are also able to bind a complementary DNA sequence to form a duplex structure. In this report, we describe a strategy for designing aptamer-based fluorescent reporters that function by switching structures from DNA/DNA duplex to DNA/target complex. The duplex is formed between a fluorophore-labeled DNA aptamer and a small oligonucleotide modified with a quenching moiety (denoted QDNA). When the target is absent, the aptamer binds to QDNA, bringing the fluorophore and the quencher into close proximity for maximum fluorescence quenching. When the target is introduced, the aptamer prefers to form the aptamer-target complex. The switch of the binding partners for the aptamer occurs in conjunction with the generation of a strong fluorescence signal owing to the dissociation of QDNA. Herein, we report on the preparation of several structure-switching reporters from two existing DNA aptamers. Our design strategy is easy to generalize for any aptamer without prior knowledge of its secondary or tertiary structure, and should be suited for the development of aptamer-based reporters for real-time sensing applications.

Adenosine Triphosphate↗
PubMed2003

Affinity analysis of a protein-aptamer complex using nonequilibrium capillary electrophoresis of equilibrium mixtures.

We propose a new method that allows the use of low-affinity aptamers as affinity probes in quantitative analyses of proteins. The method is based on nonequilibrium capillary electrophoresis of the equilibrium mixture (NECEEM) of a protein with its fluorescently labeled aptamer. In general, NECEEM of a protein with a fluorescently labeled aptamer generates an electropherogram with three characteristic features: two peaks and an exponential curve. Two peaks correspond to (i) the equilibrium amount of free aptamer in the equilibrium mixture and (ii) the amount of the protein-aptamer complex that remains intact at the time of detection. The exponential part is ascribed to the complex decaying during separation under nonequilibrium conditions. Simple analysis of the three features in experiments with known concentrations of the protein can be used for the determination of the equilibrium dissociation constant, Kd, of the aptamer-protein complex. Similar analysis of the three features in the experiment with unknown concentration of the protein and known Kd value allows the determination of the protein concentration. In this proof-of-principle work, the NECEEM method was applied to the analysis of thrombin using a fluorescein-labeled aptamer under the conditions at which the protein-aptamer complex completely decayed during the separation. We demonstrated that, despite the decay, as few as 4 x 10(6) molecules of the protein could be detected with NECEEM without sacrificing the accuracy. This sensitivity is comparable with that reported by others for the aptamer-based equilibrium method. Thus, the proposed NECEEM-based method allows the use of aptamers for highly sensitive affinity analysis of proteins even when protein-aptamer complexes are unstable.

Affinity Labels↗