Single Ribonucleotide Research Articles

Toward the Combinatorial Selection of Chemically Modified DNAzyme RNase A Mimics Active Against all-RNA Substrates

The convenient use of SELEX and related combinatorial methods of in vitro selection provides a formidable gateway for the generation of DNA enzymes, especially in the context of improving their potential as gene therapeutic agents. Here, we report on the selection of DNAzyme 12-91, a modified nucleic acid catalyst adorned with imidazole, ammonium, and guanidinium groups that provide for efficient M(2+)-independent cleavage of an all-RNA target sequence (kobs = 0.06 min(-1)). While Dz12-91 was selected for intramolecular cleavage of an all-RNA target, it surprisingly cleaves a target containing a lone ribocytosine unit with even greater efficiency (kobs = 0.27 min(-1)) than Dz9-86 (kobs = 0.13 min(-1)). The sequence composition of Dz12-91 bears a marked resemblance to that of Dz9-86 (kobs = 0.0014 min(-1) with an all-RNA substrate) that was selected from the same library to cleave a target containing a single ribonucleotide. However, small alterations in the sequence composition have a profound impact on the substrate preference and catalytic properties. Indeed, Dz12-91 displays the highest known rate enhancement for the M(2+)-independent cleavage of all-RNA targets. Hence, Dz12-91 represents a step toward the generation of potentially therapeutically active DNAzymes and further underscores the usefulness of modified triphosphates in selection experiments.

RNase H2 roles in genome integrity revealed by unlinking its activities.

Ribonuclease H2 (RNase H2) protects genome integrity by its dual roles of resolving transcription-related R-loops and ribonucleotides incorporated in DNA during replication. To unlink these two functions, we generated a Saccharomyces cerevisiae RNase H2 mutant that can resolve R-loops but cannot cleave single ribonucleotides in DNA. This mutant definitively correlates the 2–5 bp deletions observed in rnh201Δ strains with single rNMPs in DNA. It also establishes a connection between R-loops and Sgs1-mediated replication reinitiation at stalled forks and identifies R-loops uniquely processed by RNase H2. In mouse, deletion of any of the genes coding for RNase H2 results in embryonic lethality, and in humans, RNase H2 hypomorphic mutations cause Aicardi–Goutières syndrome (AGS), a neuroinflammatory disorder. To determine the contribution of R-loops and rNMP in DNA to the defects observed in AGS, we characterized in yeast an AGS-related mutation, which is impaired in processing both substrates, but has sufficient R-loop degradation activity to complement the defects of rnh201Δ sgs1Δ strains. However, this AGS-related mutation accumulates 2–5 bp deletions at a very similar rate as the deletion strain.

Expanding DNAzyme Functionality through Enzyme Cascades with Applications in Single Nucleotide Repair and Tunable DNA-Directed Assembly of Nanomaterials.

Many biological functions require two or more enzymes working together in cascades. While many examples of protein and RNA enzyme cascades are known, few enzyme cascades containing solely DNAzymes have been reported. Herein we demonstrate the combination of an 8-17 DNAzyme with RNA cleavage activity and an E47 DNAzyme with DNA ligation activity to achieve a new function of single ribonucleotide repair in DNA while maintaining the integrity of the original DNA sequence, which is difficult for a single DNAzyme to achieve. In addition, this method is applied to modify the sequences of DNA strands immobilized on the surface of nanoparticles to control the DNA-directed assembly selectively and sequentially. Such an approach can be applied to other DNAzymes with different activities to expand the functions of DNAzymes and the scope of their applications.

Ribonucleotide incorporation, proofreading and bypass by human DNA polymerase δ

In both budding and fission yeast, a large number of ribonucleotides are incorporated into DNA during replication by the major replicative polymerases (Pols α, δ and ɛ). They are subsequently removed by RNase H2-dependent repair, which if defective leads to replication stress and genome instability. To extend these studies to humans, where an RNase H2 defect results in an autoimmune disease, here we compare the ability of human and yeast Pol δ to incorporate, proofread, and bypass ribonucleotides during DNA synthesis. In reactions containing nucleotide concentrations estimated to be present in mammalian cells, human Pol δ stably incorporates one rNTP for approximately 2000 dNTPs, a ratio similar to that for yeast Pol δ. This result predicts that human Pol δ may introduce more than a million ribonucleotides into the nuclear genome per replication cycle, an amount recently reported to be present in the genome of RNase H2-defective mouse cells. Consistent with such abundant stable incorporation, we show that the 3′-exonuclease activity of yeast and human Pol δ largely fails to edit ribonucleotides during polymerization. We also show that, like yeast Pol δ, human Pol δ pauses as it bypasses ribonucleotides in DNA templates, with four consecutive ribonucleotides in a DNA template being more problematic than single ribonucleotides. In conjunction with recent studies in yeast and mice, this ribonucleotide incorporation may be relevant to impaired development and disease when RNase H2 is defective in mammals. As one tool to investigate ribonucleotide incorporation by Pol δ in human cells, we show that human Pol δ containing a Leu606Met substitution in the polymerase active site incorporates 7-fold more ribonucleotides into DNA than does wild type Pol δ.

In vitro reconstitution of RNA primer removal in Archaea reveals the existence of two pathways

Using model DNA substrates and purified recombinant proteins from Pyrococcus abyssi, I have reconstituted the enzymatic reactions involved in RNA primer elimination in vitro. In my dual-labelled system, polymerase D performed efficient strand displacement DNA synthesis, generating 5'-RNA flaps which were subsequently released by Fen1, before ligation by Lig1. In this pathway, the initial cleavage event by RNase HII facilitated RNA primer removal of Okazaki fragments. In addition, I have shown that polymerase B was able to displace downstream DNA strands with a single ribonucleotide at the 5'-end, a product resulting from a single cut in the RNA initiator by RNase HII. After RNA elimination, the combined activities of strand displacement DNA synthesis by polymerase B and flap cleavage by Fen1 provided a nicked substrate for ligation by Lig1. The unique specificities of Okazaki fragment maturation enzymes and replicative DNA polymerases strongly support the existence of two pathways in the resolution of RNA fragments.

Enzymatic Removal of Ribonucleotides from DNA Is Essential for Mammalian Genome Integrity and Development

SummaryThe presence of ribonucleotides in genomic DNA is undesirable given their increased susceptibility to hydrolysis. Ribonuclease (RNase) H enzymes that recognize and process such embedded ribonucleotides are present in all domains of life. However, in unicellular organisms such as budding yeast, they are not required for viability or even efficient cellular proliferation, while in humans, RNase H2 hypomorphic mutations cause the neuroinflammatory disorder Aicardi-Goutières syndrome. Here, we report that RNase H2 is an essential enzyme in mice, required for embryonic growth from gastrulation onward. RNase H2 null embryos accumulate large numbers of single (or di-) ribonucleotides embedded in their genomic DNA (>1,000,000 per cell), resulting in genome instability and a p53-dependent DNA-damage response. Our findings establish RNase H2 as a key mammalian genome surveillance enzyme required for ribonucleotide removal and demonstrate that ribonucleotides are the most commonly occurring endogenous nucleotide base lesion in replicating cells.

Involvement of Ser94 in RNase HIII from Chlamydophila pneumoniae in the recognition of a single ribonucleotide misincorporated into double-stranded DNA

We recently provided the first report that RNase HIII can cleave a DNA-rN1-DNA/DNA substrate (rN1, one ribonucleotide) in vitro. In the present study, mutagenesis analyses and molecular dynamics (MD) simulations were performed on RNase HIII from Chlamydophila pneumoniae AR39 (CpRNase HIII). Our results elucidate the mechanism of ribonucleotide recognition employed by CpRNase HIII, indicating that the G95/K96/G97 motif of CpRNase HIII represents the main surface interacting with single ribonucleotides, in a manner similar to that of the GR(K)G motif of RNase HIIs. However, CpRNase HIII lacks the specific tyrosine required for RNase HII to recognize single ribonucleotides in double-stranded DNA (dsDNA). Interestingly, MD shows that Ser94 of CpRNase HIII forms a stable hydrogen bond with the deoxyribonucleotide at the (5')RNA–DNA(3') junction, moving this nucleotide away from the chimeric ribonucleotide. This movement appears to deform the nucleic acid backbone at the RNA–DNA junction and allows the ribonucleotide to interact with the GKG motif. Based on the inferences drawn from MD simulations, biochemical results indicated that Ser94 was necessary for catalytic activity on the DNA-rN1-DNA/DNA substrate; mutant S94V could bind this substrate but exhibited no cleavage. Mismatches opposite the single ribonucleotide misincorporated in dsDNA inhibited cleavage by CpRNase HIII to varying degrees but did not interfere with CpRNase/substrate binding. Further MD results implied that mismatches impair the interaction between Ser94 and the deoxyribonucleotide at the RNA–DNA junction. Consequently, recognition of the misincorporated ribonucleotide was disturbed. Our results may help elucidate the distinct substrate-recognition properties of different RNase Hs.

Solution Structure of the Dickerson DNA Dodecamer Containing a Single Ribonucleotide

Ribonucleotides are frequently incorporated into DNA during replication. They are recognized and processed by several cellular enzymes, and their continued presence in the yeast nuclear genome results in replicative stress and genome instability. Thus, it is important to understand the effects of isolated ribonucleotide incorporation on DNA structure. With this goal in mind, we describe the nuclear magnetic resonance structure of the self-complementary Dickerson dodecamer sequence [d(CGC)rGd(AATTCGCG)](2) containing two symmetrically positioned riboguanosines. The absence of an observable H(1)-H(2) scalar coupling interaction indicates a C3'-endo conformation for the ribose. Longer-range structural perturbations resulting from the presence of the ribonucleotide are limited to the adjacent and transhelical nucleotides, while the global B-form DNA structure is maintained. Because crystallographic studies have indicated that isolated ribonucleotides promote global B → A transitions, we also performed molecular modeling analyses to evaluate the structural consequences of higher ribonucleotide substitution levels. Increasing the ribonucleotide content increased the minor groove width toward values more similar to that of A-DNA, but even 50% ribonucleotide substitution did not fully convert the B-DNA to A-DNA. Comparing our structure with the structure of an RNase H2-bound DNA supports the conclusion that, as with other DNA-protein complexes, the DNA conformation is strongly influenced by the interaction with the protein.

Ribonucleotide Discrimination and Reverse Transcription by the Human Mitochondrial DNA Polymerase

During DNA synthesis, DNA polymerases must select against ribonucleotides, present at much higher levels compared with deoxyribonucleotides. Most DNA polymerases are equipped to exclude ribonucleotides from their active site through a bulky side chain residue that can sterically block the 2'-hydroxyl group of the ribose ring. However, many nuclear replicative and repair DNA polymerases incorporate ribonucleotides into DNA, suggesting that the exclusion mechanism is not perfect. In this study, we show that the human mitochondrial DNA polymerase γ discriminates ribonucleotides efficiently but differentially based on the base identity. Whereas UTP is discriminated by 77,000-fold compared with dTTP, the discrimination drops to 1,100-fold for GTP versus dGTP. In addition, the efficiency of the enzyme was reduced 3-14-fold, depending on the identity of the incoming nucleotide, when it extended from a primer containing a 3'-terminal ribonucleotide. DNA polymerase γ is also proficient in performing single-nucleotide reverse transcription reactions from both DNA and RNA primer terminus, although its bypass efficiency is significantly diminished with increasing stretches of ribonucleotides in template DNA. Furthermore, we show that the E895A mutant enzyme is compromised in its ability to discriminate ribonucleotides, mainly due to its defects in deoxyribonucleoside triphosphate binding, and is also a poor reverse transcriptase. The potential biochemical defects of a patient harboring a disease mutation in the same amino acid (E895G) are discussed.

RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers

BackgroundThe polymerase chain reaction (PCR) is commonly used to detect the presence of nucleic acid sequences both in research and diagnostic settings. While high specificity is often achieved, biological requirements sometimes necessitate that primers are placed in suboptimal locations which lead to problems with the formation of primer dimers and/or misamplification of homologous sequences.ResultsPyrococcus abyssi (P.a.) RNase H2 was used to enable PCR to be performed using blocked primers containing a single ribonucleotide residue which are activated via cleavage by the enzyme (rhPCR). Cleavage occurs 5'-to the RNA base following primer hybridization to the target DNA. The requirement of the primer to first hybridize with the target sequence to gain activity eliminates the formation of primer-dimers and greatly reduces misamplification of closely related sequences. Mismatches near the scissile linkage decrease the efficiency of cleavage by RNase H2, further increasing the specificity of the assay. When applied to the detection of single nucleotide polymorphisms (SNPs), rhPCR was found to be far more sensitive than standard allele-specific PCR. In general, the best discrimination occurs when the mismatch is placed at the RNA:DNA base pair.ConclusionrhPCR eliminates the formation of primer dimers and markedly improves the specificity of PCR with respect to off-target amplification. These advantages of the assay should find utility in challenging qPCR applications such as genotyping, high level multiplex assays and rare allele detection.

Replication of ribonucleotide-containing DNA templates by yeast replicative polymerases

The major replicative DNA polymerases of S. cerevisiae (Pols α, δ, and ɛ) incorporate substantial numbers of ribonucleotides into DNA during DNA synthesis. When these ribonucleotides are not removed in vivo, they reside in the template strand used for the next round of replication and could potentially reduce replication efficiency and fidelity. To examine if the presence of ribonucleotides in a DNA template impede DNA synthesis, we determined the efficiency with which Pols α, δ, and ɛ copy DNA templates containing a single ribonucleotide. All three polymerases can replicate past ribonucleotides. Relative to all-DNA templates, bypass of ribo-containing templates is slightly reduced, to extents that depend on the identity of the ribo and the sequence context in which it resides. Bypass efficiencies for Pols δ and ɛ were increased by increasing the dNTP concentrations to those induced by cellular stress, and in the case of Pol ɛ, by inactivating the 3′-exonuclease activity. Overall, ribonucleotide bypass efficiencies are comparable to, and usually exceed, those for the common oxidative stress-induced lesion 8-oxo-guanine.

Crystal Structures of RNase H2 in Complex with Nucleic Acid Reveal the Mechanism of RNA-DNA Junction Recognition and Cleavage

SummaryTwo classes of RNase H hydrolyze RNA of RNA/DNA hybrids. In contrast to RNase H1 that requires four ribonucleotides for cleavage, RNase H2 can nick duplex DNAs containing a single ribonucleotide, suggesting different in vivo substrates. We report here the crystal structures of a type 2 RNase H in complex with substrates containing a (5′)RNA-DNA(3′) junction. They revealed a unique mechanism of recognition and substrate-assisted cleavage. A conserved tyrosine residue distorts the nucleic acid at the junction, allowing the substrate to function in catalysis by participating in coordination of the active site metal ion. The biochemical and structural properties of RNase H2 explain the preference of the enzyme for junction substrates and establish the structural and mechanistic differences with RNase H1. Junction recognition is important for the removal of RNA embedded in DNA and may play an important role in DNA replication and repair.

Structure-Specific Nuclease Activities of Pyrococcus abyssi RNase HII

Faithful DNA replication involves the removal of RNA residues from genomic DNA prior to the ligation of nascent DNA fragments in all living organisms. Because the physiological roles of archaeal type 2 RNase H are not fully understood, the substrate structure requirements for the detection of RNase H activity need further clarification. Biochemical characterization of a single RNase H detected within the genome of Pyrococcus abyssi showed that this type 2 RNase H is an Mg- and alkaline pH-dependent enzyme. PabRNase HII showed RNase activity and acted as a specific endonuclease on RNA-DNA/DNA duplexes. This specific cleavage, 1 nucleotide upstream of the RNA-DNA junction, occurred on a substrate in which RNA initiators had to be fully annealed to the cDNA template. On the other hand, a 5' RNA flap Okazaki fragment intermediate impaired PabRNase HII endonuclease activity. Furthermore, introduction of mismatches into the RNA portion near the RNA-DNA junction decreased both the specificity and the efficiency of cleavage by PabRNase HII. Additionally, PabRNase HII could cleave a single ribonucleotide embedded in a double-stranded DNA. Our data revealed PabRNase HII as a dual-function enzyme likely required for the completion of DNA replication and DNA repair.

The Structure of the Mammalian RNase H2 Complex Provides Insight into RNA·DNA Hybrid Processing to Prevent Immune Dysfunction

The mammalian RNase H2 ribonuclease complex has a critical function in nucleic acid metabolism to prevent immune activation with likely roles in processing of RNA primers in Okazaki fragments during DNA replication, in removing ribonucleotides misinserted by DNA polymerases, and in eliminating RNA.DNA hybrids during cell death. Mammalian RNase H2 is a heterotrimeric complex of the RNase H2A, RNase H2B, and RNase H2C proteins that are all required for proper function and activity. Mutations in the human RNase H2 genes cause Aicardi-Goutières syndrome. We have determined the crystal structure of the three-protein mouse RNase H2 enzyme complex to better understand the molecular basis of RNase H2 dysfunction in human autoimmunity. The structure reveals the intimately interwoven architecture of RNase H2B and RNase H2C that interface with RNase H2A in a complex ideally suited for nucleic acid binding and hydrolysis coupled to protein-protein interaction motifs that could allow for efficient participation in multiple cellular functions. We have identified four conserved acidic residues in the active site that are necessary for activity and suggest a two-metal ion mechanism of catalysis for RNase H2. An Okazaki fragment has been modeled into the RNase H2 nucleic acid binding site providing insight into the recognition of RNA.DNA junctions by the RNase H2. Further structural and biochemical analyses show that some RNase H2 disease-causing mutations likely result in aberrant protein-protein interactions while the RNase H2A subunit-G37S mutation appears to distort the active site accounting for the demonstrated substrate specificity modification.

Endoribonuclease activity of human apurinic/apyrimidinic endonuclease 1 revealed by a real-time fluorometric assay

Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional enzyme with a well-established abasic DNA cleaving activity in the base excision DNA repair pathway and in providing redox activity to several well-known transcription factors. APE1 has recently been shown to cleave at the UA, CA, and UG sites of c- myc RNA in vitro and regulates c- myc messenger RNA (mRNA) in cells. To further understand this new endoribonuclease activity of APE1, we have developed an accurate, sensitive, and rapid real-time endonuclease assay based on a fluorogenic oligodeoxynucleotide substrate with a single ribonucleotide. Using this substrate, we carried out the first kinetic analysis of APE1 endoribonuclease activity. We found that the purified native APE1 cleaves the fluorogenic substrate efficiently, as revealed by a k cat/ K m of 2.62 × 10 6 M −1 s −1, a value that is only 71-fold lower than that obtained with the potent bovine pancreatic RNase A. Ion concentrations ranging from 0.2 to 2 mM Mg 2+ promoted catalysis, whereas 10 to 20 mM Mg 2+ was inhibitory to the RNA-cleaving activity of APE1. The monovalent cation K + was inhibitory except at 20 mM, where it significantly stimulated recombinant APE1 activity. These results demonstrate rapid and specific endoribonucleolytic cleavage by APE1 and support the notion that this activity is a previously undefined function of APE1.

A novel single nucleotide polymorphism detection of a double-stranded DNA target by a ribonucleotide-carrying molecular beacon and thermostable RNase HII

Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation. SNPs are important markers that link sequence variations to phenotypic changes. Because of the importance of SNPs in the life and medical sciences, a great deal of effort has been devoted to developing accurate, rapid, and cost-effective technologies for SNP analysis. In this article, we describe a novel method for SNP genotyping based on differential fluorescence emission due to cleavage by Thermus thermophilus RNase HII (TthRNase HII) of DNA heteroduplexes containing an SNP site-specific chimeric DNA–rN 1–DNA molecular beacon (cMB). We constructed a loop sequence for a cMB that contains a single SNP-specific ribonucleotide at the central site. When the cMB probe is hybridized to a target double-stranded DNA (dsDNA), a perfect match of the cMB/DNA duplex permits efficient cleavage with TthRNase HII, whereas a mismatch in the duplex due to an SNP greatly reduces efficiency. Cleavage efficiency is measured by the incremental difference of fluorescence emission of the beacon. We show that the genotypes of 10 individuals at 12 SNP sites across a series of human leukocyte antigen (HLA) can be determined correctly with respect to conventional DNA sequencing. This novel TthRNase HII-based method offers a platform for easy and accurate SNP analysis.

Characterization of an RNA-Cleaving Deoxyribozyme with Optimal Activity at pH 5

An in vitro selection endeavor previously executed by our laboratory led to the isolation of a set of RNA-cleaving deoxyribozymes that thrive under acidic conditions [Liu, Z., Mei, S. H., Brennan, J. D., and Li, Y. (2003) J. Am. Chem. Soc. 125, 7539-7545]. One of these sequences, coined pH5DZ1, is a 100-nucleotide (nt) cis-acting enzyme that was found to exhibit high cleavage activity near pH 5. Herein, we seek to deduce the properties and sequence requirements of this enzyme. This deoxyribozyme was found to cleave a 23-nt chimeric DNA-RNA substrate, which contains a single ribonucleotide flanked by fluorophore- and quencher-modified nucleotides on each side of the cleavage junction. Extensive nucleotide deletion experiments indicated that only 42 bases within the original enzyme sequence are required for catalysis. Results from a reselection experiment further revealed that 26 of these nucleotides are absolutely conserved. In addition to sequence analysis and minimization studies, we successfully designed a trans-acting variant of this enzyme. Characterization of the cleavage products produced upon pH5DZ1-mediated RNA cleavage and analyses of possible structures of pH5DZ1 provided us with insights into the catalytic mechanism of pH5DZ1 and characteristics of deoxyribozymes that retain their activity under acidic conditions.

Structure/function Correlations in P. aeruginosa DNA Ligase LigD

The ATP-dependent DNA ligase D (LigD) performs a major role in non-homologous end-joining (NHEJ) pathway. Pseudomonas aeruginosa LigD (PaeLigD) contains a N-terminal phosphoesterase domain (PE) domain followed by a ligase domain and a C-terminal polymerase domain. The PE domain (187 residues) possesses manganese dependent phosphodiesterase and phosphomonoesterase activities as it sequentially removes the 3′-ribonucleoside from the primer strand of the RNA primer-DNA template duplex and subsequently hydrolyzes the 3′-PO4 to a 3′-OH group (1).PaeLigD-PE belongs to a class of unique 3′- end-processing enzymes as it cleaves the primer strand to a point at which a single ligatable ribonucleotide remains (2).Extensive mutagenesis studies have identified critical residues required for ribonuclease and 3′-phosphatase activities (1). Multiple active sites present in the enzyme lead to the belief that this enzyme possesses some unique motifs. However, in the absence of atomic level structural information clear structure/function correlations are lacking. We are currently working towards obtaining a high-resolution structure of PaeLigD-PE using solution NMR methods.Reference:1) Zhu, H., and Shuman, S. (2006) J. Biol. Chem. 281, 13873-13881.2) Zhu, H., and Shuman, S. (2008) J. Biol. Chem. 283, 8331-8339.

Bacterial Nonhomologous End Joining Ligases Preferentially Seal Breaks with a 3′-OH Monoribonucleotide

Many bacterial species have a nonhomologous end joining system of DNA repair driven by dedicated DNA ligases (LigD and LigC). LigD is a multifunctional enzyme composed of a ligase domain fused to two other catalytic modules: a polymerase that preferentially adds ribonucleotides to double-strand break ends and a phosphoesterase that trims 3'-oligoribonucleotide tracts until only a single 3'-ribonucleotide remains. LigD and LigC have a feeble capacity to seal 3'-OH/5'-PO(4) DNA nicks. Here, we report that nick sealing by LigD and LigC enzymes is stimulated by the presence of a single ribonucleotide at the broken 3'-OH end. The ribonucleotide effect on LigD and LigC is specific for the 3'-terminal nucleotide and is either diminished or abolished when additional vicinal ribonucleotides are present. No such 3'-ribonucleotide effect is observed for bacterial LigA or Chlorella virus ligase. We found that in vitro repair of a double-strand break by Pseudomonas LigD requires the polymerase module and results in incorporation of an alkali-labile ribonucleotide at the repair junction. These results illuminate an underlying logic for the domain organization of LigD, whereby the polymerase and phosphoesterase domains can heal the broken 3'-end to generate the monoribonucleotide terminus favored by the nonhomologous end joining ligases.

Molecular beacons for isothermal fluorescence enhancement by the cleavage of RNase HII from Chlamydia pneumoniae

This article describes a new assay for isothermal enhancement of fluorescence intensity. The assay is based on the cleavage of duplexes formed by the chimeric DNA–rN 1–DNA molecular beacon (cMB) and target DNA with Chlamydia pneumoniae RNase HII (CpRNase HII). The loop sequence of the cMB, which was designed according to the target sequence, contains a single ribonucleotide. The combination of CpRNase HII cleavage and cMB (RHMB) permitted a 90-fold increase in fluorescence intensity change compared with the hybridization reaction in the presence of the same amount of target DNA. These results indicate that the RHMB assay can enhance the fluorescence signal in real-time monitoring of the target DNA.