A catalytic 13-mer ribozyme.

A 13-mer oligoribonucleotide can act as a ribozyme for the specific self-cleavage of a 41-mer oligoribonucleotide substrate in the presence of Mg2+. The two sequences involved correspond to the self-cleavage hammerhead structure of the virusoid of lucerne transient streak virus. The Michaelis-menten kinetic parameters for the reaction were; Km 1.3 microM, Vmax 0.012 microM min-1, kcat 0.5 min-1. The 13-mer RNA is the smallest ribozyme so far reported. A DNA analogue of the 13-mer can not substitute for the RNA in the reaction.     

A circular RNA-DNA enzyme obtained by in vitro selection

A circular RNA-DNA enzyme with higher activity to target RNA cleavage and higher stability than that of the hammerhead ribozyme in the presence of RNase A was obtained by in vitro selection. The molecule is composed of a catalytic domain of 22-mer ribonucleotides derived from the hammerhead ribozyme and a fragment of 55-mer deoxyribonucleotides. The DNA fragment contains two substrate-binding domains (9-mer and 6-mer, respectively) and a "regulation domain" (assistant 40-mer DNA with 20-mer random deoxyribonucleotides sequence), which probably play the role in the regulation of flexibility and rigidity of the circular RNA-DNA enzyme. The above results suggest that the circular RNA-DNA enzyme will have a great prospect in gene-targeting therapies.     

Design and NMR analysis of HDV ribozymes for structural investigation.

Three variants of minimized hepatitis delta virus (HDV) RNA ribozyme systems (Rz-1 to approximately Rz-3) (Fig. 1) were designed on the basis of the "pseudoknot" structure model and synthesized. Rz-1 is a cis-acting ribozyme system (a cleaved form, 56-mer) in which stem IV is deleted from the active domain of genomic HDV RNA. Rz-1 was uniformly labeled with stable isotopes, 13C and 15N. The 2D-NOESY and 2D-HSQC data for Rz-1 suggest that Rz-1 forms the pseudoknot structure and G38 which is opposite to the cleavage site makes a base-pair. Rz-2 is a trans-acting ribozyme system which consists of three RNA oligomer strands (substrate: 8-mer, the cytidine residue at the cleavage site is replaced by 2'-O-methylcytidine; enzyme: 16-mer plus 35-mer). Rz-3 is a ribozyme in which the three RNA strands of Rz-2 are connected. It turns out that Rz-3 forms an inactive structure with low cleavage activity (k(obs) = 0.009) and final cleavage yield (6%). Rz-3 has the highest cleavage activity at pH 5.5. The optimal activity at acidic pH is similar to that of the wild type ribozyme. We also synthesized and examined the activity and structure of Rz-4 (designed by Perrotta and Been) which consists of two RNA strands (1).     

NMR analysis of tertiary interactions in HDV ribozymes.

Three variants of minimized hepatitis delta virus (HDV) RNA ribozyme systems designed on the basis of the "pseudoknot" model were synthesized and their tertiary interactions were analyzed by NMR spectroscopy. Rz-1 is a cis-acting ribozyme system (the cleaved form, 56-mer) in which stem IV is deleted from the active domain of genomic HDV RNA. Rz-1 was uniformly labeled with stable isotopes, 13C and 15N. Rz-2 is a trans-acting ribozyme system (substrate: 8-mer, the cytidine residue at the cleavage site is replaced by 2'-O-methylcytidine; enzyme: 16-mer plus 35-mer). Rz-2 was partially labeled with stable isotopes in guanosine residues of enzyme 35mer. Rz-4 is a trans-acting ribozyme system (substrate: 8mer, the cytidine residue at the cleavage site is replaced by 2'-O-methylcytidine; enzyme 53mer) which was designed by Perrotta and Been. Rz-4 has the same sequence and an extra loop closing stem IV. From 2D-NOESY and 2D-HSQC (except for Rz-4) spectra, it was suggested each ribozyme forms "pseudoknot" type structure in solution. Additionally, it was found that G38 of Rz-1, G28 and G29 of Rz-2 and Rz-4 form base-pairs. These novel base-pairs are observed in the crystal structure of a modified genomic HDV RNA. From temperature change experiment of Rz-2, the imino proton signal of G28 disappeared at 50 degrees C earlier than the other corresponding signals. Upon MgCl2 titration of Rz-2, this signal showed the largest shift.     

Enhancing sequence-specific cleavage of RNA within a duplex region: incorporation of 1,3-propanediol linkers into oligonucleotide conjugates of serinol-terpyridine.

The syntheses and RNA cleavage efficiencies of a new series of oligonucleotide conjugates of Cu(II)-serinol-terpyridine and 1,3-propanediol are reported. These reagents, termed ribozyme mimics, were designed such that they would yield multiple unpaired RNA residues directly opposite the site of the RNA cleavage catalyst upon ribozyme mimic-RNA duplex formation. This design effect was implemented using the 1,3-propanediol linker 3, which mimics the three-carbon spacing between the 5'- and 3'-hydroxyls of a natural nucleotide. Incorporation of one or more of these 1,3-propanediol linkers at positions directly adjacent to the serinol-terpyridine modification in the ribozyme mimic DNA strand resulted in cleavage at multiple phosphates in a complementary 31-mer RNA target sequence. The linkers effectively created artificial mismatches in the RNA-DNA duplexes, rendering the opposing RNA residues much more susceptible to cleavage via the transesterification/hydrolysis pathway. The RNA cleavage products produced by the various mimics correlated directly with the number and locations of the linkers in their DNA strands, and the most active ribozyme mimic in the series exhibited multiple turnover in the presence of excess 31-mer RNA target.     

Mutagenesis and self-ligation of the self-cleavage domain of the satellite RNA minus strand of tobacco ringspot virus and its binding to polyamines.

Several mutants for the minus strands of the self-cleaving domain of the satellite RNA of tobacco ringspot virus have been synthesized by joining chemically synthesized oligoribonucleotides with RNA ligase. Kinetic properties of the enzyme strands (50 nucleotides) against substrates (15-mer and 18-mer) were investigated. Structural properties of the unpaired part in the cleavage region were estimated from mutagenesis. The catalytic domain alone was proved to be responsible for the rejoining reaction of cleaved substrates. It was also found that the ribozyme could be divided into two strands without loss of activity. Effects of concentration of magnesium ion and polyamines on the cleavage reaction for the two-stranded ribozyme are also reported.     

The 2 A structure of helix 6 of the human signal recognition particle RNA

BACKGROUND: The mammalian signal recognition particle (SRP) is an essential cytoplasmic ribonucleoprotein complex involved in targeting signal-peptide-containing proteins to the endoplasmic reticulum. Assembly of the SRP requires protein SRP19 to bind first to helix 6 of the SRP RNA before the signal-peptide-recognizing protein, SRP54, can bind to helix 8 of the RNA. Helix 6 is closed by a GGAG tetraloop, which has been shown to form part of the SRP19-binding site. RESULTS: The high-resolution (2.0 A) structure of a fragment of human SRP RNA comprising 29 nucleotides of helix 6 has been determined using the multiple anomalous dispersion (MAD) method and bromine-labelled RNA. In the crystal the molecule forms 28-mer duplexes rather than the native monomeric hairpin structure, although two chemically equivalent 11 base pair stretches of the duplex represent the presumed native structure. The duplex has highly distorted A-RNA geometry caused by the occurrence of several non-Watson-Crick base pairs. These include a 5'-GGAG-3'/3'-GAGG-5' purine bulge (which replaces the tetraloop) and a 5'-AC-3'/3'-CA-5' tandem mismatch that, depending on the protonation state of the adenine bases, adopts a different conformation in the two native-like parts of the structure. The structure also shows the 2'3'-cyclic phosphate reaction product of the hammerhead ribozyme cleavage reaction. CONCLUSIONS: The 29-mer RNA is the first RNA structure of the human SRP and provides some insight into the binding mode of SRP19. The observed strong irregularities of the RNA helix make the major groove wide enough and flat enough to possibly accommodate an alpha helix of SRP19. The variety of non-canonical base pairs observed enlarges the limited repertoire of irregular RNA folds known to date and the observed conformation of the 2'3'-cyclic phosphate containing Ade29 is consistent with the current understanding of the hammerhead ribozyme reaction mechanism.     

Ribozymes correctly cleave a model substrate and endogenous RNA in vivo

The alpha-sarcin domain of 28 S RNA in Xenopus oocytes is attacked by several catalytic toxins (e.g. alpha-sarcin and ricin) that abolish protein synthesis. We synthesized 6 ribozymes targeted to the alpha-sarcin domain and to an oligoribonucleotide (34-mer) that mimics this domain. Sarcin ribozyme 5 (SR5) efficiently cleaved after the CUC site in the synthetic 34-mer in vitro at 50 degrees C. SR5 also cut the same site when both substrate and ribozyme were coinjected or injected separately into oocytes at 18 degrees C. Correct cleavage in vivo was shown by isolating and sequencing the large cleavage fragment. The cleavage reaction appeared to function equally well in the oocyte nucleus and cytoplasm. SR5 also correctly cleaved endogenous 28 S RNA in oocytes, although cutting was much less efficient than with alpha-sarcin. We therefore demonstrated that a ribozyme specifically cuts both a model substrate and a cellular RNA in vivo. Earlier work showed that certain injected deoxyoligonucleotides complementary to the alpha-sarcin region abolish protein synthesis. Oocyte protein synthesis was also abolished by an SR5 containing a single G----U substitution that inactivates RNA catalysis, indicating that SR5's translational suppression was perhaps due to antisense function rather than ribozyme cleavage.     

A ribozyme with DNA in the hybridising arms displays enhanced cleavage ability.

Hammerhead ribozymes cleave RNA substrates containing the UX sequence, where X = U, C or A, embedded within sequences which are complementary to the hybridising 'arms' of the ribozyme. In this study we have replaced the RNA in the hybridising arms of the ribozyme with DNA, and the resulting ribozyme is many times more active than its precursor. In turnover-kinetics experiments with a 13-mer RNA substrate, the kcat/Km ratios are 10 and 150 microM-1min-1 for the RNA- and DNA-armed ribozymes, respectively. The effect is due mainly to differences in kcat. In independent experiments where the cleavage step is rate-limiting, the DNA-armed ribozyme cleaves the substrate with a rate constant more than 3 times greater than the all-RNA ribozyme. DNA substrates containing a ribocytidine at the cleavage site have been shown to be cleaved less efficiently than their all-RNA analogues; again however, the DNA-armed ribozyme is more effective than the all-RNA ribozyme against such DNA substrates. These results demonstrate that there are no 2'-hydroxyl groups in the arms of the ribozyme that are required for cleavage; and that the structure of the complex formed by the DNA-armed ribozyme with its substrate is more favourable for cleavage than that formed by the all-RNA ribozyme and its substrate.     

Importance of specific adenosine N7-nitrogens for efficient cleavage by a hammerhead ribozyme. A model for magnesium binding.

Five modified hammerhead ribozyme/substrate complexes have been prepared in which individual adenosine N7-nitrogens have been excised. The modified complexes were chemically synthesized with the substitution of a single 7-deazaadenosine (c7A) base analogue for residues A11, A14, A26, A27, or A28. Two of the base analogues, c7A11 and c7A14, occur in a 19-mer ribozyme, while the remaining three residues, c7A26, c7A27, and c7A28, are present in a 24-mer substrate. Under stoichiometric conditions, four of the complexes are cleaved with relatively little change in rate when compared with that of the native complex. However, the relative rate for the c7A11 complex is some 35-fold slower than that of the native complex. Steady-state kinetic analyses indicate that the cleavage efficiencies, as measured by kcat/KM, for the c7A14, c7A26, c7A27, and c7A28 complexes are reduced 18-fold, 10-fold, 34-fold, and 16-fold, respectively. These reductions in cleavage efficiency are primarily a result of lower kcat values. By comparison, the cleavage efficiency of the c7A11 complex is reduced more than 200-fold relative to that of the native complex, again primarily as a result of a lower kcat value. The results suggest that the N7-nitrogen of A11 in the hammerhead ribozyme/substrate complex is critical for efficient cleavage activity. The results of the present work, in combination with those from previous reports, indicate that five critical functional groups are located within the tetrameric sequence G10A11U12G13. A preliminary model for the binding of a single magnesium cofactor to this portion of the sequence is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assessment of disparate structural features in three models of the hepatitis delta virus ribozyme.

Three models for the secondary structure of the hepatitis delta virus (HDV) antigenomic self-cleaving RNA element were tested by site-directed mutagenesis. Two models in which bases 5' to the cleavage site are paired with sequence at the 3' end of the element were both inconsistent with the data from the mutagenesis. Specifically, mutations in the 3' sequence which decrease self-cleavage activity could not be compensated by base changes in the 5' sequence as predicted by these models. The evidence was consistent with a third model in which the 3' end pairs with a portion of a loop within the ribozyme sequence to generate a pseudoknot structure. This same pairing was also required to generate higher rates of cleavage in trans with a 15-mer ribozyme, thus ruling out a proposed hammerhead-like 'axehead' model for the HDV ribozyme.     

Hairpin ribozyme specificity in vivo: a case of promiscuous cleavage

We have used differential display to address the question of ribozyme specificity in vivo. Stably transfected PC12 cells bearing either a hairpin ribozyme expression plasmid targeted to betaAPP mRNA or the vector alone were analyzed using nine different primer pairs. One of the few differentially expressed genes obtained from this screen corresponded to rat ribosomal protein L19. Steady-state levels of L19 mRNA were lower in ribozyme-transfected cells compared to either vector-transfected cells or native PC12 cells, and a sequence within the L19 message was cleaved by the betaAPP hairpin ribozyme in vitro. These data imply that sequence-specific unintended cleavage of non-target mRNAs may present a formidable problem to the use of hairpin ribozyme therapeutic agents.     

固定化锤头状ribozyme的初步研究

用共价连接方式将专一切割苜蓿夜蛾核多角体病毒多角体蛋白ｍＲＮＡ片段的锤头状ｒｉｂｏｚｙｍｅ（ｈａｍｍｅｒｈｅａｄｒｉｂｏｚｙｍｅ）固定在ＳｅｐｈａｒｏｓｅＣＬ－４Ｂ上,ｒｉｂｏｚｙｍｅ与载体之间有１９个原子的距离,研究并比较了该固定化ｒｉｂｏｚｙｍｅ与游离（非固定化）ｒｉｂｏｚｙｍｅ的底物专一性、催化活性、最适反应温度以及热和保存稳定性。讨论了固定化ｒｉｂｏｚｙｍｅ与固定化酶在性质上的相似性以及固定化ｒｉｂｏｚｙｍｅ研究在理论和应用上的可能意义。     

Site-specific modification of functional groups in genomic hepatitis delta virus (HDV) ribozyme.

Human hepatitis delta (HDV) ribozyme is one of small ribozymes, such as hammerhead and hairpin ribozymes, etc. Its secondary structure shows pseudoknot structure composed of four stems (I to IV) and three single-stranded regions (SSrA, -B and -C). The 3D structure of 3'-cleaved product of genomic HDV ribozyme provided extensive information about tertiary hydrogen bonding interactions between nucleotide bases, phosphate oxygens and 2'OHs including new stem structure P1.1. To analyze the role of these hydrogen bond networks in the catalytic reaction, site-specific atomic-level modifications (such as deoxynucleotides, deoxyribosyl-2-aminopurine, deoxyribosylpurine, 7-deaza-ribonucleotide and inosine) were incorporated in the smallest trans-acting HDV ribozyme (47-mer). Kinetic analysis of these ribozyme variants demonstrated the importance of the two W-C base pairs of P1.1 for cleavage; in addition, the results suggest that all hydrogen bond interactions detected in the crystal structure involving 2'-OH and N7 atoms are present in the active ribozyme structure. In most of the variants, the relative reduction in kobs caused by substitution of the 2'-OH group correlated with the number of hydrogen bonds affected by the substitution. However G74 and C75 may have more than one hydrogen bond involving the 2'-OH in both the trans- and cis-acting HDV ribozyme. Moreover, in variants in which N7 was deleted, kobs was reduced 5- to 15-fold, it may suggest that N7 assists in coordinating Mg2+ ions or water molecules which bind with weak affinity in the active structure.     

Structural features of target RNA molecules greatly modulate the cleavage efficiency of trans-acting delta ribozymes

The aim of this work was to shed some more light on factors influencing the effectiveness of delta ribozyme cleavage of structured RNA molecules. An oligoribonucleotide that corresponds to the 3'-terminal region X of HCV RNA and yeast tRNAPhe were used as representative RNA targets. Only a few sites susceptible to ribozyme cleavage were identified in these targets using a combinatorial library of ribozyme variants, in which the region responsible for ribozyme-target interaction was randomized. On the other hand, the targets were fairly accessible for binding of complementary oligonucleotides, as was shown by 6-mer DNA libraries and RNase H approach. Moreover, the specifically acting ribozymes cleaved the targets precisely but with unexpectedly modest efficacy. To explain these observations, six model RNA molecules were designed, in which the same seven nucleotide long sequence recognized by the delta ribozyme was always single stranded but was embedded into different RNA structural context. These molecules were cleaved with differentiated rates, and the corresponding k2 values were in the range of 0.91-0.021 min-1; thus they differed almost 50-fold. This clearly shows that cleavage of structured RNAs might be much slower than cleavage of a short unstructured oligoribonucleotide, despite full accessibility of the targeted regions for hybridization. Restricted possibilities of conformational transitions, which are necessary to occur on the cleavage reaction trajectory, seem to be responsible for these differences. Their magnitude, which was evaluated in this work, should be taken into account while considering the use of delta ribozymes for practical applications.     

Ribozyme-catalyzed dipeptide synthesis in monovalent metal ions alone

Previously we have identified a highly active ribozyme (R180, cis ribozyme) that can catalyze dipeptide synthesis using N-biotinylcaproyl-aminoacyl-adenylate anhydride (Bio-aa-5'-AMP) as its substrate. In this work, we re-engineered the cis R180 ribozyme into a 158-nt trans ribozyme (TR158) and designed a new substrate (5'-Phe-linker-20-mer). First, the metal ion requirements were examined and compared between the two ribozymes. Both R180 and TR158 ribozymes were active in Mg2+ and Ca2+ but inert with Zn2+, Cu2+, Mn2+, and Co2+. It is intriguing that both ribozymes were highly active in Li+, Na+, or K+ alone but showed very low activity with NH4+. The two ribozymes showed similar linear concentration dependence on Li+ and K+, while they displayed different dependency behavior on Mg2+. Moreover, by using the trans system, the detailed kinetic studies and pH dependent experiments were performed in either 10 mM Mg2+ or 1.0 M Li+. Analysis of kcat and Km values obtained at different pHs (6.0 to 9.0) indicated that it is the catalytic activity of the ribozyme but not the substrate binding affinity that changes significantly with pH. The slopes of the linear parts of the pH-rate plots were close to 1.0 in both Mg2+- and Li+-mediated reactions, suggesting that one proton transfer is involved in the rate-limiting step of catalysis. Overall, our results suggest that Mg2+ and Li+ function similarly in the ribozyme-catalyzed dipeptide synthesis.     

Mg2+-dependent folding of a Diels-Alderase ribozyme probed by single-molecule FRET analysis

Here, we report a single-molecule fluorescence resonance energy transfer (FRET) study of a Diels-Alderase (DAse) ribozyme, a 49-mer RNA with true catalytic properties. The DAse ribozyme was labeled with Cy3 and Cy5 as a FRET pair of dyes to observe intramolecular folding, which is a prerequisite for its recognition and turnover of two organic substrate molecules. FRET efficiency histograms and kinetic data were taken on a large number of surface-immobilized ribozyme molecules as a function of the Mg²⁺ concentration in the buffer solution. From these data, three separate states of the DAse ribozyme can be distinguished, the unfolded (U), intermediate (I) and folded (F) states. A thermodynamic model was developed to quantitatively analyze the dependence of these states on the Mg²⁺ concentration. The FRET data also provide information on structural properties. The I state shows a strongly cooperative compaction with increasing Mg²⁺ concentration that arises from association with several Mg²⁺ ions. This transition is followed by a second Mg²⁺-dependent cooperative transition to the F state. The observation of conformational heterogeneity and continuous fluctuations between the I and F states on the ~100ms timescale offers insight into the folding dynamics of this ribozyme.     

Probing the mechanisms of DEAD-box proteins as general RNA chaperones: the C-terminal domain of CYT-19 mediates general recognition of RNA

The DEAD-box protein CYT-19 functions in the folding of several group I introns in vivo and a diverse set of group I and group II RNAs in vitro. Recent work using the Tetrahymena group I ribozyme demonstrated that CYT-19 possesses a second RNA-binding site, distinct from the unwinding active site, which enhances unwinding activity by binding nonspecifically to the adjacent RNA structure. Here, we probe the region of CYT-19 responsible for that binding by constructing a C-terminal truncation variant that lacks 49 amino acids and terminates at a domain boundary, as defined by limited proteolysis. This truncated protein unwinds a six-base-pair duplex, formed between the oligonucleotide substrate of the Tetrahymena ribozyme and an oligonucleotide corresponding to the internal guide sequence of the ribozyme, with near-wild-type efficiency. However, the truncated protein is activated much less than the wild-type protein when the duplex is covalently linked to the ribozyme or single-stranded or double-stranded extensions. Thus, the active site for RNA unwinding remains functional in the truncated CYT-19, but the site that binds the adjacent RNA structure has been compromised. Equilibrium binding experiments confirmed that the truncated protein binds RNA less tightly than the wild-type protein. RNA binding by the compromised site is important for chaperone activity, because the truncated protein is less active in facilitating the folding of a group I intron that requires CYT-19 in vivo. The deleted region contains arginine-rich sequences, as found in other RNA-binding proteins, and may function by tethering CYT-19 to structured RNAs, so that it can efficiently disrupt exposed, non-native structural elements, allowing them to refold. Many other DExD/H-box proteins also contain arginine-rich ancillary domains, and some of these domains may function similarly as nonspecific RNA-binding elements that enhance general RNA chaperone activity.     

Inhibition of HIV-1 integrase by modified oligonucleotides derived from U5' LTR.

Retroviral integrase catalyzes integration of double-stranded viral DNA into the host chromosome by a process that has become an attractive target for drug design. In the 3' processing reaction, two nucleotides are specifically cleaved from both 3' ends of viral DNA yielding a 5' phosphorylated dimer (pGT). The resulting recessed 3' hydroxy groups of adenosine provide the attachment sites to the host DNA in the strand transfer reaction. Here, we studied the effect of modified double-stranded oligonucleotides mimicking both the unprocessed (21-mer oligonucleotides) and 3' processed (19-mer oligonucleotides) U5 termini of proviral DNA on activities of HIV-1 integrase in vitro. The inhibitions of 3' processing and strand transfer reactions were studied using 21-mer oligonucleotides containing isopolar, nonisosteric, both conformationally flexible and restricted phosphonate internucleotide linkages between the conservative AG of the sequence CAGT, and using a 21-mer oligonucleotide containing 2'-fluoroarabinofuranosyladenine. All modified 21-mer oligonucleotides competitively inhibited both reactions mediated by HIV-1 integrase with nanomolar IC50 values. Our studies with 19-mer oligonucleotides showed that modifications of the 3' hydroxyl significantly reduced the strand transfer reaction. The inhibition of integrase with 19-mer oligonucleotides terminated by (S)-9-(3-hydroxy-2-phosphonomethoxypropyl)adenine, 9-(2-phosphonomethoxyethyl)adenine, and adenosine showed that proper orientation of the 3' OH group and the presence of the furanose ring of adenosine significantly influence the strand transfer reaction.     

Development of non-electrophoretic assay method for DNA ligases and its application to screening of chemical inhibitors of DNA ligase I

A new rapid assay method for DNA ligases has been developed, which allows direct quantification of enzyme activity without using the traditional polyacrylamide gel electrophoretic technique. In this method, the 5'-biotinylated nicked duplex was used as a substrate for the ligase reaction, in which the 5'-end of the first oligonucleotide (19-mer) on the nicked strand is biotinylated and the second oligonucleotide (20-mer) on the same strand is labeled with radioactive 32P at the 5'-end. After ligation of the biotinylated 19-mer oligonucleotide into the second oligonucleotide with the reaction of DNA ligases, the biotinylated 19-mer oligonucleotide is converted into the radioactive 39-mer oligonucleotide. The ligase reaction products were heat-denatured to release both ligated and unligated biotinylated oligonucleotides. The biotinylated oligonucleotides were then captured on a streptavidin-coated microtiter plate and counted. The results obtained using this method correlated very well with those from the standard assay method using electrophoresis. Using this assay method, we were able to screen a chemical library and identify new DNA ligase inhibitors structurally related to resorcinol, which has growth inhibitory effects on the human breast cancer cell, MCF-7. The method described here is anticipated to be very useful for screening DNA ligase inhibitors from chemical libraries.