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Razvan Nutiu

Publications and source records attributed to Razvan Nutiu.

13 recordsLinked to original sources
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↗
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

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↗
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↗
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↗
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↗
PubMed2002

Tripartite molecular beacons.

Molecular beacons (MBs) are hairpin-like fluorescent DNA probes that have single-mismatch detection capability. Although they are extremely useful for many solution-based nucleic acid detections, MBs are expensive probes for applications that require the use of a large number of different DNA probes due to the high cost and tedious procedures associated with probe synthesis and purification. In addition, since both ends of MB probes are covalently modified with chromophores, they do not offer the flexibility for fluorophore change and the capability for surface immobilization through free DNA ends. In this report, we describe an alternative form of MB, denoted tripartite molecular beacon (TMB), that may help overcome these problems. A TMB uses an unmodified oligodeoxyribonucleotide that forms a MB-like structure with two universal single-stranded arms to bring on a universal pair of oligodeoxyribonucleotides modified separately with a fluorophore and a quencher. We found that TMBs are as effective as standard MBs in signaling the presence of matching nucleic acid targets and in precisely discriminating targets that differ by a single nucleotide. TMBs have the necessary flexibility that may make MBs more affordable for various nucleic acid detection applications.

Base Sequence↗