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Fluorescence-Signaling Nucleic Acid-Based Sensors

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It is widely known that two single-stranded nucleic acids with complementary sequences have the inherent ability to form Watson-Crick duplex structures. The simplicity and sequence-specificity of duplex structure formation, the high chemical stability of a duplex, and the convenience of automated synthesis have made DNA oligonucleotides an ideal choice as probes for the detection of nucleic acids. Recently developed in vitro selection techniques permit creation of DNA and RNA “aptamers“ that are capable of binding a wide variety of nonnucleic acid targets with high affinity and specificity. Aptamers have considerably broadened the utility of nucleic acids as probes for detection of biological and nonbiological targets. In vitro selection also allows generation of artificial ribozymes (catalytic RNAs) and deoxyribozymes (catalytic DNAs) with desirable functions. Aptamers, ribozymes, and deoxyribozymes have become increasingly valuable molecular tools in the form of switches and sensors. Unfortunately, binding or catalytic actions by these switches and sensors do not usually lead to an easily detectable signal, and the lack of a facile reporting method could substantially reduce their value. To facilitate the exploitation of nucleic acid switches and sensors for detection-related applications, many recent studies have explored fluorescence signaling as a convenient approach for the reporting of binding and catalytic events. This chapter is devoted to the discussion of these efforts. The reporter molecules to be described include molecular beacons, signaling aptamers, and signaling ribozymes and deoxyribozymes.

Introduction

Since the report of the first biosensor in 1967,1,2 tremendous progress has been made in the biosensor field. By conventional definition, a biosensor refers to an analytical device consisting of two important components: a molecular recognition element (MRE) for target (analyte) detection and a transducer that physically reports the MRE-analyte interaction. In such a device, the MRE acts as an affinity probe that specifically seeks an analyte from a complex biological sample for binding. Many naturally occurring and artificial substances can be explored as MREs, and the choice of a specific MRE for a particular application is often dependent on the nature of the analyte and the availability of MREs.3 The widely utilized MREs include natural receptors,4 protein enzymes and antibodies,5 cells,6 organelles,7 tissues8 and molecularly imprinted polymers.9

The suitability of nucleic acids as specific MREs for the detection of DNA and RNA targets is clear due to their inherent ability to form Watson-Crick duplexes. In a broad sense, Southern blotting10 can be regarded as a primitive biosensor that uses nucleic acids as MREs. Over the last decade, the exploitation of nucleic acids as MREs has been significantly extended beyond the detection of nucleic acid targets, thanks to the ground-breaking discoveries that certain single-stranded DNA or RNA molecules termed “aptamers“ can form defined tertiary structures for binding nonnucleic acid targets.11,12 From the extensive studies reported to date, it is increasingly apparent that DNA and RNA aptamers represent a new class of versatile MREs for the detection of a broad scope of nonnucleic acid analytes, including proteins and metabolites.13,14

Aptamers offer several distinct advantages as MREs. Aptamers are isolated in vitro, and the targets for aptamer creation can be any compounds including toxins. Once an aptamer is identified, it can be prepared either by automated DNA synthesis at very low cost and with high batch-consistency (in the case of DNA) or by simple in vitro transcription (in the case of RNA). Moreover, it is easy to modify aptamers to introduce various reactive groups, affinity tags, or reporting moieties that are required for biosensing applications. Finally, DNA aptamers have a long shelf life and usually retain activity following a denaturation-renaturation process.

Of course, molecular recognition elements can only be formulated into a biosensing device if the MRE-analyte recognition event can be transduced into an easily detectable signal. One important step in developing biosensing systems that exploit aptamers as MREs is to establish effective methods that report an aptamer-analyte binding event. Signal transduction can be achieved through electrochemical, mechanical, piezoelectric, or fluorescent means, and each of these methods have been applied to aptamers.15-18 Among these methods, fluorescence signaling is highly desirable because of the ease of detection, the availability of diverse measurement methods, and the existence of a diverse collection of fluorophores and quenchers suitable for nucleic acid modification.19 In comparison to more traditional radiolabeling approaches that are often exploited in nucleic acid assays, fluorescence detection confers several advantages. First, fluorescent substrates are not subjected to the signal decay inherent to radioactive substrates. Second, fluorescence is amenable to real-time or continuous assays, in contrast to the laborious and time-consuming discontinuous assays necessary when working with radioactive substrates. Third, fluorescence-based platforms lend themselves well to preexisting fiber-optic and DNA microarray technologies.20,21 Therefore, various strategies for the design of effective fluorescence-signaling aptamers are being actively pursued by many research groups.

Similar to aptamers, catalytic nucleic acids (ribozymes and deoxyribozymes) can be generated de novo by in vitro selection from an extraordinarily large population of single-stranded RNA or DNA molecules.22 Although no catalytic DNA has been found in nature, many research laboratories around the world have isolated hundreds of DNA sequences that facilitate more than a dozen chemical transformations, often involving nucleic acid substrates.23-26 For example, DNA enzymes have been isolated that cleave DNA27 and RNA,28,29 ligate RNA30 and phosphorylate DNA.31 Ribozymes and deoxyribozymes may have limited value for direct application as MREs because of the relatively small scope of substrates and/or cofactors. However, the recent development of allosteric nucleic acid enzymes—RNA and DNA molecules consisting of both an aptamer domain and a catalytic domain—has made catalytic RNA and DNA very useful as part of MREs.32 As a result, increasing efforts have been focused on developing fluorescence-based sensor molecules made of ribozymes and deoxyribozymes.

In this chapter, we discuss recent progress in the development of novel fluorescent nucleic acid probes as MREs for the detection of nucleic acid targets and nonnucleic acid targets. We first describe molecular beacons—hairpin-shaped fluorescent DNA probes that are extremely useful for nucleic acid detection. This is followed by a review of the engineering of signaling aptamers. Finally, we discuss examples of engineering fluorescence-signaling ribozymes and deoxyribozymes identified either by in vitro selection or by post-selection modifications.

Molecular Beacons for Nucleic Acid Detection

Because standard nucleic acid bases are inherently nonfluorescent, chemical modifications are required to make them fluorescent. The simplest fluorescence-signaling method for nucleic acid detection is the use of an organic chromophore that can bind to nucleic acids with accompanying changes in its fluorescence properties. Many compounds such as ethidium bromide and Hoechst 33342 have very weak intrinsic fluorescence in solution but display significantly enhanced emission when bound to duplex DNA or RNA. Nucleic acids can also be made fluorescent by the use of nucleobase analogs such as 2-aminopurine (for adenine) or isoxanthopterin (for guanine). The most attractive method for making DNA or RNA fluorescent is usually the covalent attachment of fluorophores or quenchers directly onto the nucleic acid. An external fluorophore can be joined either to an end of a nucleic acid chain or coupled onto an internal base. The modification can be carried out during the solid-phase synthesis of DNA or RNA using fluorophore-containing phosphoramidite monomers; during the enzymatic synthesis of DNA or RNA using fluorophore-modified nucleoside triphosphates; or through post-synthetic coupling using facile reactions and simple procedures.

A new group of fluorescent DNA probes, known as molecular beacons (MB), have become increasingly popular. MBs are single-stranded DNA probes doubly modified with a fluorophore (F) and a quencher (Q) at the two ends of the DNA strand and are useful for DNA or RNA detection with a signaling mechanism that is illustrated in Figure 1.33 The sequence of each MB is designed to form a stem-loop structure. The stem of a MB is formed between two complementary segments of usually 5-7 nucleotides (nt) in length that are located at the opposite ends of the DNA strand. The loop sequence is specifically designed to be complementary to the sequence of a target DNA or RNA molecule. Because of these design elements, an MB can adopt two structural states, the “open“ state and the “closed“ state. In the closed state, the fluorophore is located in close proximity to the quencher, and fluorescence is not observed. However, when the target is present, the MB adopts the open state due to the formation of more stable loop-target duplex. Because in the open state the quencher is spatially separated from the fluorophore, the emission of fluorescence is restored. The loop size and stem strength of an MB can be fine-tuned such that only a target with a fully complementary sequence can efficiently induce the transition from the closed state to the open state. Therefore, MBs offer very high sequence specificity in target recognition and can usually discriminate sequences differing in a single nucleotide. Because MBs have a build-in fluorescent switch, target detection can be performed in real time and does not require a lengthy procedure to separate the target-probe complex from free probes. Furthermore, several MBs labeled with different fluorophores can be used to conduct a multiplexed assay for the analysis of different targets in one solution.34 The fluorescence reporting of MBs is very sensitive, and fluorescence enhancements upon target binding of up to two orders of magnitude have been reported.35

Figure 1. Standard molecular beacons.

Figure 1

Standard molecular beacons. A fluorophore (F) and a quencher (Q) are covalently linked to the two ends of the hairpin-shaped DNA probe. The formation of loop-target duplex structure causes the separation of F from Q and the generation of a fluorescence (more...)

MBs can be designed or utilized in several variations, each offering advantages for particular applications. For example, dual-fluorophore labeled MBs offer the option of monitoring emission intensities at two different wavelengths, indicative of engagement or disengagement of FRET (fluorescence resonance energy transfer) between the two fluorophores (fig. 2).36 The two beacons/one target FRET approach (fig. 3) reduces the chance of false positives that may result from factors other than target-probe duplex formation.37 The two MBs are designed to target the neighboring sequences of the same DNA or RNA molecule. The fluorophores on the two MBs are chosen for their ability to engage each other for FRET. When both MBs are opened by the target, the two fluorophores are brought in close proximity to activate FRET. However, if MBs are open due to nonspecific interactions or degraded by a contaminating nuclease, FRET is absent because the two fluorophores are not located near each other.

Figure 2. Molecular beacons with two fluorophores.

Figure 2

Molecular beacons with two fluorophores. These molecular beacons offer the options of monitoring the disappearance of FRET from the acceptor fluorophore and appearance of fluorescence signal from the donor fluorophore.

Figure 3. Two-MB/one-target design.

Figure 3

Two-MB/one-target design. This approach enhances the detection specificity and reduces false positive signaling.

Wavelength-shifting MBs (wsMBs; fig. 4) are triply labeled with two fluorophores and one quencher.38 The two fluorophores are chosen to be a FRET pair; therefore the excitation of the first fluorophore (called the harvester) will lead to the emission of the second fluorophore (emitter), provided that the quencher is physically distant due to target binding. wsMBs offer enhanced detection sensitivity relative to the simpler approach of Figure 2 due to an increased Stokes shift. Most attractively, several wsMBs can be constructed with a common harvester and different emitters for a multiplexed assay that uses monochromatic excitation light.

Figure 4. Wavelength-shifting molecular beacons.

Figure 4

Wavelength-shifting molecular beacons. These molecular beacons can be used for multiplexed assays using a fixed excitation wavelength, if two (or more) beacons are used for two (or more) targets.

A standard MB can also be reformulated into a tripartite molecular beacon (TMB) assembly (fig. 5).39 In TMBs, the fluorophore and the quencher are not covalently attached to the ends of the hairpin probe; instead they are linked to the ends of two new short oligonucleotides denoted FDNA (containing a fluorophore) and QDNA (having a quencher). FDNA and QDNA are designed to hybridize with the two extended arms of the unmodified hairpin probe and convert it into an MB-like assembly (fig. 5). Since a FDNA/QDNA combination can be used as a universal pair of labeling probes, the TMB approach is particularly attractive for applications that require a large number of MBs. In other words, the FDNA/QDNA pair can be used universally with many MBs since they (and their complementary sequences) interact with neither the stem that closes the MB nor the target recognition sequence.

Figure 5. Tripartite molecular beacons.

Figure 5

Tripartite molecular beacons. F and Q are linked to two separate short oligonucleotides (denoted FDNA and QDNA), which hybridize with corresponding single-stranded arms extended beyond the short hairpin stem. This figure is adapted from ref. .

Ideally, a MB should produce a large fluorescence signal upon target binding. Because the signal generation is a de-quenching process in the case of Figure 1, an appropriate quencher must be chosen for a particular fluorophore to reduce the level of background fluorescence (i.e., the fluorescence intensity prior to the target addition). Some small organic molecules such as DABCYL and Black Hole Quenchers are popular due to their high quenching efficiency for most fluorophores, as well as their thermal stability and photo-stability. Other nonorganic quenchers have also been described. For example, a 1.4-nm diameter gold nanoparticle is a highly effective quencher for many fluorophores in the MB context.40 Gold can also be used as a surface for DNA immobilization, and in such a setting there is no need for a separate quencher, because the gold surface acts as a quencher.41 The main drawback of nanogold is its thermal instability, making it impossible to use such a quencher in applications that involve high-temperature steps, such as the polymerase chain reaction (PCR).

MBs have been studied for many nucleic acid detection and reporting applications. For example, MBs have widely been used as DNA-reporting probes for PCR. A PCR cycle consists of three temperature steps: denaturation (94°C), annealing (50-60°C) and extension (72°C). MBs are usually designed to function during the annealing step. When the DNA amplification target (amplicon) is present in low amounts, most MB molecules stay in the closed state and emit low fluorescence. When the amplicon concentration increases during PCR, more MB molecules bind to their targets and become highly fluorescent. The use of MBs as reporter molecules in PCR offers two attractive features. First, the monitoring of DNA amplicons can be achieved in real time without any post-PCR sample manipulation. Second, MB-based detection methods are highly sequence-specific because MBs will only produce a fluorescence signal when the amplicon contains the sequence complementary to the loop segment of the hairpin structure.

One important application for MBs is as PCR probes for human allele genotyping. For example, MBs have been used as elegant tools to screen the human population for susceptibility to HIV-1 infection. It is known that individuals with a 32-nt deletion in the β-chemokine receptor 5 gene in homozygous chromosomes (mutant homozygous), with a 32-nt deletion in only one chromosome (mutant heterozygous), and with no deletion (wild-type homozygous) are largely resistant, partially resistant, and susceptible to HIV-1 infection, respectively. Kostrikis et al exploited this fact in the design of the two MBs shown in Figure 6. The first MB (MB1, labeled with a fluorophore that emits green fluorescence) targets the deletion sequence for binding, while the second MB (MB2, labeled with a fluorophore that emits red fluorescence) targets the two stretches of nucleotides sandwiching the deletion sequence for binding.42 When the DNA from an individual is used as a PCR template and the two MBs are used as detection probes, increases in fluorescence from MB1 only, from MB2 only and from both MB1 and MB2 indicate wild-type homozygous, mutant homozygous and mutant heterozygous, respectively.

Figure 6. Molecular beacons for genotyping.

Figure 6

Molecular beacons for genotyping. A pair of molecular beacons labeled with two different fluorophores permits identification of alleles with or without deletion mutations.

The power of PCR, coupled with real-time and sequence-specific detection by MBs, allows the fast and accurate characterization of minute amounts of biologically relevant nucleic acid material. To date, MBs have been extensively used for DNA, RNA and pathogenic detection; many examples can be found at http://www.molecularbeacons.com/Publications.html. MBs have also been exploited in bioanalytical assays designed to characterize DNA- or RNA-binding proteins;43 report enzymatic processes such as cleavage of DNA or RNA by nucleases;44,45 monitor in vitro transcription;46 and report on rolling circle amplification.47 Moreover, as we will discuss next, the molecular beacon concept has been adapted to the design of signaling aptamers and signaling catalytic nucleic acids.

Signaling Aptamers

The development of in vitro selection techniques has led to the generation of DNA and RNA aptamers that bind a wide variety of nonnucleic acid targets that include proteins and metabolites. Like any other nucleic acid molecules, aptamers are inherently nonfluorescent, and therefore post-selection modifications with external fluorophores must be performed to convert aptamers into fluorescence-signaling reporters.

An ideal rational design strategy for engineering signaling aptamers should have three key features. First, the method should be easy to apply to any given aptamer regardless of its size and structural properties. This is important because aptamers have variable sizes and vastly different secondary and tertiary structures. Some aptamers do not have an easily determinable secondary structure, and the tertiary structures of most aptamers are not readily available. Second, the method should be capable of designing signaling aptamers with large signaling magnitudes, fast responses, and real-time signaling capabilities. Signaling aptamers that exhibit large fluorescence enhancements upon target binding increase the sensitivity and accuracy of the corresponding assays. The real-time reporting capability allows rapid sample measurements and permits demanding applications such as high-throughput screening. Third, the method should be adaptable for the design of a signaling aptamer from any existing aptamer without altering its affinity and specificity.

One signaling aptamer design strategy is to attach a single fluorophore at different locations on the aptamer and test each construct for altered spectroscopic properties upon target binding. Jhaveri et al first reported this approach in 2000 (fig. 7).48 They used two existing ATP aptamers—one RNA and one DNA—with defined tertiary structures as model examples. Several aptamers, each with a fluorophore attached at a location distant from the target-binding site, were examined. Five modified aptamers did not show fluorescence changes in response to the addition of ATP, and the remaining two registered a 25-45% increase in fluorescence intensity upon ATP addition. The failures might have been caused by disruption of the correct structural folding of the aptamer as a result of fluorophore attachment, or by minimal alteration of the emission of the attached fluorophore when the aptamer binds to the target molecule. Aptamers with improved signaling magnitudes were reported in a later study where bis-pyrene was used as the fluorescent tag.49

Figure 7. Single-fluorophore signaling aptamers by rational design.

Figure 7

Single-fluorophore signaling aptamers by rational design. Labeling an aptamer with a fluorophore at a location where a substantial structural reorganization upon target binding induces a change in the fluorescence property of the attached fluorophore. (more...)

Jhaveri et al also investigated the direct acquisition of signaling aptamers through in vitro selection (fig. 8). To do so, they constructed a pool of random-sequence RNAs each containing one or a few fluoresceinated uridines.50 After several rounds of selective amplification using ATP as the binding target, a few aptamers that act as real-time reporters for ATP were obtained. The best signaling aptamer exhibited approximately two-fold fluorescence intensity increase at the saturating ATP concentration. Interestingly, the fluorescein label on the aptamer could be substituted by other fluorophores without affecting the aptamer's target-binding affinity and specificity. It is noteworthy that their selection also generated several aptamers that failed to register significant fluorescence enhancement upon target binding, suggesting that even the selection approach offers no guarantee regarding the generation of aptamers that are effective fluorescent reporters.

Figure 8. Single-fluorophore signaling aptamers by in vitro selection.

Figure 8

Single-fluorophore signaling aptamers by in vitro selection. Signaling aptamers are generated de novo by SELEX using a random-sequence DNA or RNA library in which each DNA or RNA molecule is labeled with one or a few fluorophores. This figure is adapted (more...)

As discussed earlier, MBs usually exhibit large signaling magnitudes upon target binding. Although standard molecular beacons are only useful for nucleic acid detection, several groups have attempted to adapt the same principle for the design of signaling aptamers. The resulting constructs are often termed aptamer beacons. Hamaguchi et al made the most straightforward adaptation (fig. 9A) by adding a few nucleotides onto the 5'-end of a small thrombin DNA aptamer to engage the 3'-end of the aptamer into a hairpin structure.51 When the protein target is absent, the aptamer beacon forms the closed-state structure in which fluorescence is quenched. In the presence of thrombin, the aptamer beacon forms the target-complexed structure in which the fluorophore and the quencher are spatially separated, resulting in a larger fluorescence signal. A two-chain aptamer beacon approach has also been reported.52 In this design, Yamamoto et al split an HIV Tat protein-binding RNA aptamer into two molecules, one of which was formulated into a molecular beacon (fig. 9B). In the absence of Tat, the two RNA molecules do not interact, and the solution has low fluorescence intensity. When Tat is introduced, the molecular beacon and the other half of the aptamer assemble into a single unit for Tat binding. The dissociation of the hairpin structure is accompanied by an increase of fluorescence intensity.

Figure 9. A) Signaling aptamers by one-chain molecular beacon approach.

Figure 9

A) Signaling aptamers by one-chain molecular beacon approach. An aptamer sequence is extended so that it can be formulated into a molecular beacon. B) Signaling aptamers by two-chain molecular beacon approach. An aptamer is split into two chains, one (more...)

Other groups have explored fluorescence quenching as an alternate way to track target-aptamer recognitions, where the detection depends on loss (rather than gain) of fluorescence signal upon binding of the target to the aptamer. Stojanovic et al used a two-chain-assembly approach in the design of two aptamer reporters, one that binds cocaine and the other for ATP recognition (fig. 9C).53 In the absence of the target, the two chains of the aptamer (one labeled with a quencher and the other with a fluorophore) do not associate strongly, and therefore the fluorescence intensity of the solution is high. Introduction of the target promotes the assembly of the two chains for target binding, resulting in fluorescence quenching. Moreover, the two aptamer reporters, each labeled with a different fluorophore, can function to some extent in the same solution, suggesting the development of aptamer-based multiplexed detection. Another fluorescence-quenching thrombin signaling aptamer, as illustrated in Figure 10, was later described by Li et al.54 It is known that the two ends of the anti-thrombin DNA aptamer are located next to each other in the folded guanine-quartet complex structure. Based on this folding property, Li et al designed a signaling aptamer by appending a fluorophore-quencher pair to the two ends of the aptamer sequence.54 The resulting aptamer was able to perform real-time reporting of thrombin by fluorescence quenching. Similarly, a DNA aptamer for the B chain of platelet-derived growth factor (PDGF) labeled with a fluorophore-quencher pair at its termini shows fluorescence quenching upon addition of PDGF.55

Figure 10. Signaling aptamers based on tertiary folding.

Figure 10

Signaling aptamers based on tertiary folding. This strategy works with aptamers whose tertiary folding brings the two ends of the aptamer chain into close proximity. This figure is adapted from ref. .

Our group has recently described a different approach for the design of signaling aptamers by exploiting the common ability of each aptamer to form two completely different structural forms: a relatively weak duplex structure with a complementary DNA oligonucleotide and a stronger complexed structure with the selected target (fig. 11). First, we attach a fluorophore onto the aptamer and a quencher onto the complementary oligonucleotide that can anneal with part of the aptamer sequence. In the absence of the target, the oligonucleotides form a low-fluorescence double-helix structure. When the target is present, the aptamer switches from the duplex structure to the complexed structure, releasing the quencher-containing antisense oligonucleotide into solution. Spatial separation of the fluorophore and quencher generates a highly intense fluorescent signal. We called these designed signaling aptamers “structureswitching signaling aptamers“ to emphasize the common structure-switching mechanism. Two aptamers, one for ATP and one for thrombin, with considerably different sizes, tertiary structures, and affinities for their targets, were successfully transformed into structure-switching signaling aptamers.56

Figure 11. Structure-switching signaling aptamers.

Figure 11

Structure-switching signaling aptamers. A modified aptamer is configured into a low-fluorescence duplex structure state with the fluorophore-containing aptamer and quencher-bearing QDNA. Target binding releases QDNA, leading to a highly fluorescent complexed (more...)

Recently, a novel fluorophore-tagging technique has been described.57 Fluorescamine (FCM) is a fluorogenic compound that is known to react very rapidly with amine groups. If an aptamer is synthesized with one or a few 2'-amine substituted nucleotides, formation of the usually compact aptamer-target complex structure will reduce the FCM-tagging activity of the amine groups. Several variants of the anti-ATP DNA aptamer with 2'-amine substituted nucleotides were constructed and tested for FCM-tagging activities in the presence of and absence of ATP. One construct showed considerably reduced reactivity when bound to ATP. This construct was then transformed into a signaling aptamer by appending a new fluorophore (F1) on the end of the DNA sequence capable of engaging FCM (F2) for FRET (fig. 12). In the presence of ATP, FCM was not able to react efficiently with the 2' amine group on the aptamer, and therefore no FRET was observed.

Figure 12. Signaling aptamers by in situ labeling.

Figure 12

Signaling aptamers by in situ labeling. Target-aptamer complex formation prevents the attachment of the donor fluorophore to an aptamer sequence labeled with the acceptor fluorophore, resulting in the lack of FRET.

Signaling Ribozymes and Deoxyribozymes

Many signaling ribozymes and deoxyribozymes have also been described in recent years. Fluorescently labeled ribozymes and deoxyribozymes have two significant advantages relative to other signaling approaches. First, the catalytic activity of a signaling enzyme with a built-in fluorescent switch can be followed conveniently by the change of fluorescence intensity in real time. If the enzyme activity is regulated by a cofactor or an effector, real-time signaling allows facile monitoring of the presence of the regulating molecule. Second, fluorescence signaling creates new opportunities for exploring nucleic acid enzymes for innovative applications, some of which are discussed below.

Many of the reported studies that involve signaling ribozymes and deoxyribozymes share two general features. First, all of the signaling systems are based on the concepts of FDQ (fluorescence de-quenching) or FRET. Second, all of the nucleic acid enzymes employed to date are RNA-cleaving ribozymes or deoxyribozymes. Both FDQ and FRET entail the use of two chromophores that must be brought together for fluorescence quenching or FRET; alternatively, the chromophores must be separated for FDQ or disappearance of FRET. For the latter purpose, a cleavage-based system is a logical choice. The dual labels can be attached onto the catalytic system in two ways: both labels can be attached to the substrate at opposite sides of the cleavage site, or one label can be placed on the substrate and the other on the enzyme at a location that is close to the first label. In both cases, the cleavage will lead to the separation of the two labels and either FDQ or the disappearance of FRET.

FRET and FDQ were initially applied for real-time monitoring of the kinetics of naturally occurring RNA-cleaving ribozymes, such as the hammerhead and hairpin ribozymes. These studies were performed either in vitro58-61 or in vivo.62 Jenne et al extended this approach to search for inhibitors of hammerhead ribozyme activity, using a modified substrate containing a fluorophore at one end and a quencher at the other (fig. 13).63 When the hammerhead ribozyme is active, cleavage of the substrate separates the fluorophore from the quencher, which leads to an increase in fluorescence intensity via FDQ. However, if the ribozyme is inhibited, the solution will have constant fluorescence intensity. A library of 96 small molecules was tested, and 16 compounds were found to modulate the activity of the hammerhead ribozyme considerably, with Ki varying from 0.1 to 100 μM.

Figure 13. Screening ribozyme inhibitors using signaling ribozymes.

Figure 13

Screening ribozyme inhibitors using signaling ribozymes. The binding of an inhibitor to a ribozyme shuts off both the catalytic activity of the ribozyme and its signaling capability.

Signaling ribozymes and deoxyribozymes have also been designed as sensors for metal ions, nucleic acids, and proteins. In principle, any metal-ion-dependent ribozyme or deoxyribozyme can be used as a selective metal ion sensor if it requires a particular divalent metal ion as the cofactor for function and is significantly less active in the presence of other metal ions. Li et al explored this principle and successfully designed a lead (Pb2+) sensor using an RNA-cleaving DNAzyme named 17E.64 17E is a sequence variant of a small DNA enzyme known as 8-17 that has been independently isolated several times by in vitro selection.29,65-67 17E was found to exhibit a strong catalytic activity in the presence of Pb2+ but significantly reduced activity when other divalent metal ions were supplied. By labeling the substrate strand with a fluorophore (F) and the DNAzyme with a quencher (Q), 17E can conveniently report the presence of Pb2+ in solution (fig. 14). Prior to the enzymatic action, the substrate binds tightly to the DNAzyme, bringing the fluorophore and the quencher into close proximity for efficient fluorescence quenching. Upon Pb2+ -promoted enzymatic action, the DNAzyme cleaves the single ribonucleotide linkage embedded in an otherwise all-DNA substrate. The two separated substrate fragments no longer anneal tightly to the DNAzyme, resulting in the increase of fluorescence. The catalytic system was ≈80-fold more responsive to Pb2+ than other divalent metal ions, with a detection range of 10 nM to 4 μM Pb2+.64,68

Figure 14. A deoxyribozyme-based Pb2+ sensor.

Figure 14

A deoxyribozyme-based Pb2+ sensor. The sensor is constructed using an RNA-cleaving DNAzyme. Upon addition of Pb2+ ions, the DNAzyme is activated and cleaves the single ribonucleotide linkage (Ar, adenosine ribonucleotide) in the substrate strand. The (more...)

The specificity of deoxyribozyme-based metal ion biosensors can be enhanced by in vitro selection with the incorporation of a negative selection strategy.69 To eliminate the sequences that are active in the presence of nontarget metal ions, the DNA library is first exposed to a complex “soup“ of metal ions that lacks the desired target ion. Next, the sequences that are active in the presence of the metal ion of interest are isolated. Exposing the selected DNA populations to decreased concentrations of the desired metal ion during the later stages of in vitro selection can also increase the affinity of the enzyme for the metal ion cofactor.

In addition to the fluorogenic form of the 17E DNAzyme for metal ion sensing, Liu and Lu have also created an elegant colorimetric lead biosensor that can be monitored visually.68 Unreacted enzyme-substrate complexes mediate the assembly of gold nanoparticles into large colored aggregates. Upon enzyme activation, the assembly of gold nanoparticles dissociates with a concurrent change in color. The sensitivity of the system is tunable to a detection range of 10-200 μM Pb2+ through introduction of inactive enzyme strands into the gold nanoparticle network.

The intrinsic ability of nucleic acids to form duplex structures has been exploited in many nucleic acid detection systems, such as the molecular beacon designs described above. Two elegant fluorescence-signaling systems—catalytic molecular beacons70 and DzyNA-PCR71— have recently been developed that ingeniously incorporate the catalytic activities of DNA for nucleic acid detection. Catalytic molecular beacons (CMBs) combine the concepts and advantages of both molecular beacons and DNAzymes (fig. 15).70 In the first reported example, the previously selected RNA-cleaving deoxyribozyme named 12E was reengineered to contain three intricately linked elements: a hairpin-shaped structural motif (labeled as “Molecular beacon module“ in fig. 15), a DNAzyme module, and a separate dual-labeled substrate that competes with the MB module for DNAzyme binding. In the absence of the target oligonucleotide, the stem of the MB module forms a highly stable hairpin structure with one of the substrate binding arms of the DNAzyme, thereby preventing the full interaction of the DNAzyme with its substrate. When the target oligonucleotide is added, duplex formation between the target and the MB module disrupts the inhibitory hairpin stem, freeing the previously unavailable substrate-binding arm and restoring enzymatic activity. The substrate is end-labeled with two fluorophores for FRET, and cleavage at the single RNA site abolishes the FRET signal and regenerates the emission signal of the donor fluorophore. The above design strategy is applicable to any RNA-cleaving DNAzyme that binds its substrate through duplex formation. One significant advantage of CMBs over standard molecular beacons is their inherent signal-amplification ability, because DNAzymes are capable of cleaving substrates with multiple turnover.70

Figure 15. A catalytic molecular beacon.

Figure 15

A catalytic molecular beacon. The system uses an MB module to transduce the molecular recognition of an oligonucleotide target to a change in fluorescence intensity, through deoxyribozyme-mediated cleavage of a dual-chromophore labelled substrate. This (more...)

In addition to their roles as catalytic platforms for nucleic acid detection, CMBs have been used in molecular computing. Using CMBs as a starting point, Stojanovic et al have made sets of several oligonucleotide-sensitive DNAzymes into logic networks able to receive oligonucleotide “inputs“ and perform such functions as AND, XOR, and NOT with fluorescent readouts.72 They also created DNAzyme networks able to receive inputs and make decisions, so-called half-adder functions.73,74 Recently, they used a combination of logic gates made of CMBs regulated by one, two or even three oligonucleotides for the creation of MAYA, an invincible tic-tac-toe DNA player.74 Although these DNAzyme-based computing devices may never rival silicon-based computing, their greatest potential may be in designing “smart“ therapeutics that are able both to diagnose and to treat biological problems.73

Hartig et al75-77 have reported several cases of oligonucleotide-regulated ribozymes. For example, they have developed signaling hammerhead ribozymes regulated by an attached RNA aptamer that is responsive to a protein target (fig. 16).75 When the protein is absent, the aptamer acts as the antisense oligonucleotide to disrupt substrate binding to the ribozyme, which inhibits its activity. When the protein is present, the aptamer abandons the ribozyme, removing the antisense suppression effect. The activation of the enzyme leads to the cleavage of the fluorogenic substrate and generation of fluorescence. Such aptamer-regulated ribozymes were utilized for the screening of small-molecule inhibitors for a protein target. Three small molecules that bind HIV-1 REV—coumermycin A1, nosiheptide and patulin—were identified by this approach.

Figure 16. A protein-responsive allosteric signaling ribozyme.

Figure 16

A protein-responsive allosteric signaling ribozyme. The substrate recognition portion of an enzyme is changed to anneal with a portion of the appended aptamer in the absence of the protein target. Binding of the protein to the aptamer disrupts the inhibitory (more...)

The invention of DzyNA-PCR, a technique that is capable of monitoring PCR reactions in real time, is another elegant example of exploiting the catalytic activity of DNA for nucleic acid detection (fig. 17).71 In this technique, one PCR primer is designed to contain a sequence complementary to the RNA-cleaving 10-23 DNA enzyme. Upon PCR amplification, the corresponding sense strand (containing the 10-23 DNAzyme sequence) is generated, which can then cleave a substrate modified with a fluorophore-quencher pair. An increase in fluorescence accompanies the amplification of the target DNA as increasing amounts of the active 10-23 DNA enzyme are generated. The DzyNA-PCR system is able to quantitate target DNA (i.e., the PCR template) over six orders of magnitude, from 101 to 107 molecules.71 This system also eloquently illustrates the robust nature of DNAzymes, because 10-23 can work under buffer conditions and temperatures that are different from those used in the in vitro selection process that first discovered this DNAzyme.71

Figure 17. DzyNA-PCR for amplification and detection of specific nucleic acid sequences.

Figure 17

DzyNA-PCR for amplification and detection of specific nucleic acid sequences. The DzyNA primer contains a target-specific sequence in addition to the antisense sequence of an RNA-cleaving DNAzyme. During amplification, a DNAzyme (as part of the sense (more...)

One significant limitation associated with the end-labeled fluorogenic substrates is high background fluorescence.70 The inflexibility of modifying preexisting DNAzymes limits improvement in this area, because introduction of fluorescent labels closer to the cleavage site usually inactivates DNAzymes. Our group has recently conducted two in vitro selection studies to isolate efficient, fluorescence-signaling, RNA-cleaving deoxyribozymes that cleave a chimeric RNA/DNA substrate in which a lone RNA linkage is flanked immediately on either side by a fluorophore and a quencher (fig. 18A). Prior to the catalytic action of the deoxyribozyme, the fluorophore and quencher are located within a short distance of each other, resulting in very efficient fluorescence quenching. Upon cleavage of the RNA linkage and subsequent product dissociation, the fluorophore moves away from the quencher, leading to intense fluorescence signaling. In the initial study,78 we created an effective RNase denoted DET22-18 (fig. 18B). DET22-18 can perform multiple turnover catalysis with a kcat of ≈7 min-1. Following this study, we created a series of RNA-cleaving signaling deoxyribozymes with various pH optima in the range from pH 3-8 (fig. 19).79 The deoxyribozymes were selected first by subjecting the DNA population to multiple rounds of in vitro selection at pH 4. Subsequently, evolution into five different pH-dependent deoxyribozyme pools was achieved by conducting five parallel paths of in vitro evolution at pH 3, 4, 5, 6 and 7. The dominant species from each pH pool were designated as pH3DZ1, pH4DZ1, pH5DZ1, pH6DZ1, and pH7DZ1 (fig. 19A). Interestingly, the four DNA enzymes selected at pH 3-6 displayed maximum catalytic rates at or near their selection pH (fig. 19B). The only catalytic DNA for which no pH optimum was detected was pH7DZ1, whose catalytic rate constant increased from pH 5.5 to 8.0. Most of the DNA enzymes exhibited relatively large catalytic rate constants with kobs values ranging between 0.2 and 1.3 min-1; only pH3DZ1 was significantly less efficient, with a kobs of 0.023 min-1. All five DNA enzymes were evaluated for their ability to produce a fluorescence enhancement upon catalysis. The existence of a series of signaling deoxyribozymes covering 5 pH units broadens the utility of signaling DNA enzymes for practical biosensing applications. Further engineering of these catalytic DNA species into target-reporting probes is currently underway.

Figure 18. Signaling deoxyribozymes with closely located fluorophore and quencher pairs.

Figure 18

Signaling deoxyribozymes with closely located fluorophore and quencher pairs. A) Catalytic scheme. B) Sequence and secondary structure of a trans-acting signaling deoxyribozyme denoted DET22-18. This figure is adapted from ref. .

Figure 19. Signaling deoxyribozymes with different pH profiles.

Figure 19

Signaling deoxyribozymes with different pH profiles. A) The sequences of five signaling deoxyribozymes identified by in vitro selection. The lowercase letters represent nucleotides that are not essential for catalysis. B) Normalized catalytic rates versus (more...)

Outlook

The abundant examples presented above illustrate diverse strategies for designing nucleic acid switches and sensors that conveniently transduce an event of molecular recognition and/or catalysis into an easily detectable fluorescence signal. Most studies conducted to date have aimed to demonstrate proof of concept and used artificial targets in “clean“ solutions. The next and more challenging step is to adapt these concepts and probes to more realistic applications. For example, one can imagine the construction of a multiplexed system for simultaneously tracking key components of a living cell (mRNAs, proteins, and metabolites) under a specific set of conditions or under the influence of external stimuli. This would allow the study of cellular processes on a more comprehensive scale, offering more of a global view rather than the limited information acquired by one approach or one technology at a time. Although the application of fluorescent nucleic acid probes for studying living systems is still at an incipient stage, the limited results obtained so far are exciting and promising. For example, Matsuo and colleagues have conducted a study aimed to visualize mRNA in living cells using MBs. They designed a MB targeting the mRNA for basic fibroblast growth factor (bFGF) and successfully visualized the mRNA of bFGF in a human cell line.80 Similarly, MBs have been successfully applied to detect vav protooncogene mRNA in K562 human leukemia cells81 and β-actin mRNA in single living kangaroo rat kidney cells.82 Recently, Bratu et al has conducted a study to follow the transport and localization of mRNA in Drosophila oocytes.37 By microinjecting a nuclease-resistant MB for a target sequence of the oskar mRNA and following the hybridization kinetics in real time, mRNA transport was visualized from its birthplace to its final destination. To date, there are no research papers that describe the use of signaling aptamers or signaling nucleic acid enzymes for in vivo imaging of small molecules or proteins. However, aptamers have been shown to function inside cells.83 Therefore, we anticipate that ongoing efforts will lead to the creation of signaling aptamers and signaling nucleic acid enzymes for in vivo applications.

Perhaps one of the most valuable uses of signaling aptamers and signaling nucleic acid enzymes is in the field of drug discovery. As discussed in this chapter, signaling aptamers and allosteric nucleic acid enzymes can be engineered to report both protein enzymes and small-molecule metabolites. Therefore, these nucleic acid probes may be extremely useful as fluorescent reporters in screening assays for drug discovery, and their potential is currently under investigation in several academic and industrial labs. Two different concepts have been developed to date. The first approach is based on an affinity competition for a particular protein between a nucleic acid enzyme and a small molecule. For example, if the protein binds an allosteric signaling ribozyme, the resulting catalytic activity generates a fluorescent signal. In contrast, if the protein prefers to bind to a small molecule such as a potential drug, then the signaling ribozyme remains inactive, and no fluorescence can be detected.75 The second approach is designed to detect the activity of an enzyme by monitoring the consumption of the reactants or formation of the products. Consider a reaction A→B mediated by an enzyme E. If a signaling aptamer has different affinities for A and B, then the enzymatic transformation of A to B is transduced into a change in fluorescence intensity. Consequently, compounds that inhibit E can be identified by monitoring the lack of fluorescence change.84 The same approach can be used for the case of metabolite-responsive allosteric DNA or RNA enzymes.85 Although the reported examples are currently at the proof-of-concept stage, the encouraging results hold promise for extensive use of signaling aptamers and signaling nucleic acid enzymes in high-throughput drug discovery in the years to come.

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