Chimeric DNA-RNA Research Articles

Effect of the Phosphoryl Guanidine Modification in Chimeric DNA-RNA crRNAs on the Activity of the CRISPR-Cas9 System In Vitro.

Currently, the CRISPR-Cas9 system serves as a prevalent tool for genome editing and gene expression regulation. Its therapeutic application is limited by off-target effects that can affect genomic integrity through nonspecific, undesirable changes in the genome. Various strategies have been explored to mitigate the off-target effects. Many approaches focus on modifying components of the system, namely, Cas9 and guide RNAs, to enhance specificity. However, a common challenge is that methods aiming to increase specificity often result in a significant reduction in the editing efficiency. Here, we introduce a novel approach to modifying crRNA to balance CRISPR-Cas9 specificity and efficiency. Our approach involves incorporating nucleoside modifications, such as replacing ribo- to deoxyribonucleosides and backbone modifications, using phosphoryl guanidine groups, specifically 1,3-dimethylimidazolidin-2-ylidene phosphoramidate. In this case, within the first 10 nucleotides from the 5' crRNA end, phosphodiester bonds are substituted with phosphoryl guanidine groups. We demonstrate that crRNAs containing a combination of deoxyribonucleosides and single or multiple phosphoryl guanidine groups facilitate the modulation of CRISPR-Cas9 system activity while improving its specificity in vitro.

Split activator of CRISPR/Cas12a for direct and sensitive detection of microRNA

CRISPR/Cas12a-based nucleic acid assays have been increasingly used for molecular diagnostics. However, most current CRISPR/Cas12a-based RNA assays require the conversion of RNA into DNA by preamplification strategies, which increases the complexity of detection. Here, we found certain chimeric DNA-RNA hybrid single strands could activate the trans-cleavage activity of Cas12a, and then discovered the activating effect of split ssDNA and RNA when they are present simultaneously. As proof of concept, split nucleic acid-activated Cas12a (SNA-Cas12a) strategy was developed for direct detection of miR-155. By adding a short ssDNA to the proximal end of the crRNA spacer sequence, we realized the direct detection of RNA targets using Cas12a. With the assistance of ssDNA, we extended the limitation that CRISPR/Cas12a cannot be activated by RNA targets. In addition, by taking advantage of the programmability of crRNA, the length of its binding to DNA and RNA was optimized to achieve the optimal efficiency in activating Cas12a. The SNA-Cas12a method enabled sensitive miR-155 detection at pM level. This method was simple, rapid, and specific. Thus, we proposed a new Cas12a-based RNA detection strategy that expanded the application of CRISPR/Cas12a.

Highly specific chimeric DNA-RNA-guided genome editing with enhanced CRISPR-Cas12a system

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system is composed of a Cas12a effector that acts as a DNA-cleaving endonuclease and a crispr RNA (crRNA) that guides the effector to the target DNA. It is considered a key molecule for inducing target-specific gene editing in various living systems. Here, we improved the efficiency and specificity of the CRISPR-Cas12a system through protein and crRNA engineering. In particular, to optimize the CRISPR-Cas12a system at the molecular level, we used a chimeric DNA-RNA guide chemically similar to crRNA to maximize target sequence specificity. Compared with the wild-type (wt)-Cas12a system, when using enhanced Cas12a system (en-Cas12a), the efficiency and target specificity improved on average by 2.58 and 2.77 times, respectively. In our study, when the chimeric DNA-RNA-guided en-Cas12a effector was used, the gene-editing efficiency and accuracy were simultaneously increased. These findings could contribute to highly accurate genome editing, such as human gene therapy, in the near future.

Chimeric DNA-RNA Guide RNA Designs.

CRISPR-associated nuclease (Cas) has been widely applied to modify the genomes of various cell types. As RNA-guided endonucleases, Cas enzymes can target different genomic sequences simply by changing the guide sequence of the CRISPR RNA (crRNA) or single guide RNA (sgRNA). Recent studies have demonstrated that DNA-RNA chimeric crRNA or sgRNA can efficiently guide the Cas9 protein for genome editing with reduced off-target effects. This chapter aims to describe a procedure for using chimeric RNA to modify the genomes of mammalian cells.

Bacterial Genome Containing Chimeric DNA-RNA Sequences.

Almost five decades ago Crick, Orgel, and others proposed the RNA world hypothesis. Subsequent studies have raised the possibility that RNA might be able to support both genotype and phenotype, and the function of RNA templates has been studied in terms of evolution, replication, and catalysis. Recently, we engineered strains of E. coli in which a large fraction of 2'-deoxycytidine in the genome is substituted with the modified base 5-hydroxymethyl-2'-deoxycytidine. We now report the generation of mutant strains derived from these engineered bacteria that show significant (∼40-50%) ribonucleotide content in their genome. We have begun to characterize the properties of these chimeric genomes and the corresponding strains to determine the circumstances under which E. coli can incorporate ribonucleotides into its genome and herein report our initial observations.

Atomic resolution structure of a chimeric DNA-RNA Z-type duplex in complex with Ba(2+) ions: a case of complicated multi-domain twinning.

The self-complementary dCrGdCrGdCrG hexanucleotide, in which not only the pyrimidine/purine bases but also the ribo/deoxy sugars alternate along the sequence, was crystallized in the presence of barium cations in the form of a left-handed Z-type duplex. The asymmetric unit of the P21 crystal with a pseudohexagonal lattice contains four chimeric duplexes and 16 partial Ba(2+) sites. The chimeric (DNA-RNA)2 duplexes have novel patterns of hydration and exhibit a high degree of discrete conformational disorder of their sugar-phosphate backbones, which can at least partly be correlated with the fractional occupancies of the barium ions. The crystals of the DNA-RNA chimeric duplex in complex with Ba(2+) ions and also with Sr(2+) ions exhibit complicated twinning, which in combination with structural pseudosymmetry made structure determination difficult. The structure could be successfully solved by molecular replacement in space groups P1 and P21 but not in orthorhombic or higher symmetry and, after scrupulous twinning and packing analysis, was refined in space group P21 to an R and Rfree of 11.36 and 16.91%, respectively, using data extending to 1.09 Å resolution. With the crystal structure having monoclinic symmetry, the sixfold crystal twinning is a combination of threefold and twofold rotations. The paper describes the practical aspects of dealing with cases of complicated twinning and pseudosymmetry, and compares the available software tools for the refinement and analysis of such cases.

Imaging an Expanding Molecular Robot World Using Super-Accuracy Single-Molecule Fluorescence Microscopy

We recently demonstrated the concept of molecular robotics with a synthetic DNA-based nanowalker, dubbed a “spider” composed of a streptavidin protein “body” attached to three biotinylated DNA enzyme legs, along a one-dimensional track of chimeric DNA-RNA substrates positioned on a DNA origami (1,2). By cleaving its substrates, the spider weakens the binding energy between its legs and previously visited sites, resulting in a biased, processive, random walk towards fresh substrate. Additional components are now being incorporated into the spider world to increase its versatility and complexity in behavior. For example, we are implementing a second spider that walks on a different substrate. This spider can be differentially controlled from our original nanowalker via changes in buffer conditions that favor one spider over the other and vice versa. This advance will allow us to devise a controlled “spider race” towards a common goal post; depending on the predetermined winner, a payload will either be released from the goal or not, behaving as an XOR gate. By fluorophore labeling the different components, we aim to characterize this spider race at the single molecule level using super-accuracy total internal reflection fluorescence microscopy (TIRFM). 1. Lund, K., Manzo, A.J., Dabby, N., Michelotti, N., Johnson-Buck, A., Nangreave, J., Taylor, S., Pei, R., Stojanovic, M.N., Walter, N.G., Winfree, E., and Yan, H. (2010) Molecular robots guided by prescriptive landscapes. Nature 465, pp. 206–210. 2. Michelotti, N., de Silva, C., Johnson-Buck, A.E., Manzo, A.J., Walter, N.G. (2010) A bird's eye view: tracking slow nanometer-scale movements of single molecular nano-assemblies. Methods Enzymol. 475, pp. 121–148.

TGF-β1 mRNAに対するDNA-RNAキメラ型リボザイムによる被嚢胞性腹膜硬化症の遺伝子治療の開発

TGF-β1 mRNAに対するDNA-RNAキメラ型リボザイムによる被嚢胞性腹膜硬化症の遺伝子治療の開発

Single-Molecule Tracking of Nanorobots on Pseudo-One-Dimensional DNA Origami Tracks

A synthetic molecular nanorobot dubbed a spider composed of a streptavidin protein body bound to three biotinylated 8-17 deoxyribozyme-bearing completes the programmed task of traversing a predetermined DNA origami track. The track is assembled by folding and holding in place a long single-stranded DNA molecule into a two-dimensional landscape by using hundreds of short oligonucleotides (staples) and hybridizing chimeric DNA-RNA substrates to specific staples to realize a pseudo-one-dimensional track. Spiders are designed to undergo a biased random walk as their legs repeatedly cleave oligonucleotide substrates on a track, causing the legs to more readily dissociate from the cleaved product and progress towards the uncleaved substrate. We image Cy3-labeled spiders and Cy5-labeled origami using total internal reflection fluorescence microscopy (TIRFM). By fitting Gaussian functions to the imaged point spread functions, we are able to monitor with nanometer spatial precision the real-time motion of the spiders along DNA origami tracks.

Use of Chimeric DNA-RNA Primers in Quantitative PCR for Detection of Ehrlichia canis and Babesia canis

To overcome the problem of nonspecific by-products in quantitative PCR (qPCR) assays, we constructed DNA-RNA chimeric primers and evaluated their use in the detection and quantification of the Ehrlichia canis 16S rRNA, Babesia canis Hsp70, and canine beta-actin genes. Several RNA bases were incorporated into specific positions in the DNA primers, while no RNA stretches were allowed. qPCR reactions were carried out without preamplification steps. This resulted in decreased formation of undesirable by-products and a 10-fold increase in assay sensitivity.

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.

Development of a Chimeric DNA-RNA Hammerhead Ribozyme Targeting SARS Virus

Objective: Severe acute respiratory syndrome (SARS) is a severe pulmonary infectious disease caused by a novel coronavirus. To develop an effective and specific medicine targeting the SARS-coronavirus (CoV), a chimeric DNA-RNA hammerhead ribozyme was designed and synthesized using a sequence homologous with the mouse hepatitis virus (MHV). Method: Chimeric DNA-RNA hammerhead ribozyme targeting MHV and SARS-CoV were designed and synthesized.To confirm its activity, in vitro cleavage reactions were performed with the synthesized ribozyme. Effects of the chimeric ribozyme were evaluated on multiplication of MHV. Effects of the chimeric ribozyme on expression of SARS-CoV were evaluated in cultured 3T3 cells. Result: The synthetic ribozyme cleaved the synthetic target MHV and SARS-CoV RNA into fragments of predicted length. The chimeric DNA-RNA hammerhead ribozyme targeting SARS-CoV significantly inhibited multiplication of MHV in DBT cells by about 60%. The chimeric DNA-RNA hammerhead ribozyme targeting SARS-CoV significantly inhibited the expression of SARS-CoV RNA in 3T3 cells transfected with the recombinant plasmid. The chimeric DNA-RNA ribozyme targeting SARS-CoV significantly inhibited MHV viral activity and expression of recombinant SARS RNA in vitro. Conclusion: These findings indicate that the synthetic chimeric DNA-RNA ribozyme could provide a feasible treatment for SARS.

Chimeric DNA–RNA hammerhead ribozyme targeting transforming growth factor-β1 mRNA ameliorates renal injury in hypertensive rats

Transforming growth factor (TGF)-beta is a critical factor in the progression of renal injury, regardless of the primary etiology. Such injury is characterized by glomerular sclerosis and tubulointerstitial fibrosis. To develop a ribozyme-based therapy for progressive renal diseases, we examined the effects of chimeric DNA-RNA hammerhead ribozyme targeting TGF-beta1 mRNA on glomerulosclerosis in salt-loaded, stroke-prone spontaneously hypertensive rats (SHR-SP) and salt-sensitive Dahl (Dahl-S) rats. The chimeric DNA-RNA ribozyme to TGF-beta1 was delivered by polyethylenimine to cultured mesangial cells from SHR-SP in vitro and to glomeruli in SHR-SP in vivo. The chimeric ribozyme reduced expression of TGF-beta1 mRNA and protein, which was accompanied by inhibition of expression of extracellular matrix molecules such as fibronectin and collagen type I in mesangial cells from SHR-SP in vitro. One intraperitoneal injection of 200 microg of chimeric DNA-RNA ribozyme to TGF-beta1 in vivo markedly ameliorated thickening of capillary artery walls and glomerulosclerosis in salt-loaded SHR-SP and Dahl-S rats without a reduction in blood pressure. The chimeric ribozyme reduced expression of TGF-beta1 and connective tissue growth factor (CTGF) mRNAs in renal cortex in salt-loaded Dahl-S rats. Chimeric ribozyme to TGF-beta1 significantly reduced levels of protein in urine in the Dahl-S rats. These results suggest that chimeric DNA-RNA ribozyme to TGF-beta1 may be useful as a gene therapy for progressive tissue injury in a wide variety of renal diseases, including hypertensive nephrosclerosis.

Inhibition of Growth of Human Gingival Fibroblasts by Chimeric DNA‐RNA Hammerhead Ribozyme Targeting Transforming Growth Factor‐β1

Transforming growth factor (TGF)-beta1 is involved in the pathogenesis of both drug-induced gingival overgrowth and hereditary gingival fibromatosis. Ribozymes enzymatically cleave target mRNAs and are expected to be utilized as the basis of novel nucleic acid-based therapies. We designed a chimeric DNA-RNA ribozyme targeting TGF-beta1 mRNA and examined its effect on growth of gingival fibroblasts in culture. Chimeric DNA-RNA hammerhead ribozyme with sequence complementary to the loop structure of human TGF-beta1 mRNA was used. We evaluated transfer of the chimeric ribozyme by hemagglutinating virus of Japan (HVJ)-envelope into cultured human gingival fibroblasts in vitro and rat gingival tissues in vivo. We then examined effects of the chimeric ribozyme to TGF-beta1 on proliferation and DNA synthesis in human gingival fibroblasts. We also examined effects of the chimeric ribozyme to TGF-beta1 on expression of TGF-beta1, type IV collagens, and fibronectin mRNAs and expression of TGF-beta1 protein in human gingival fibroblasts. Chimeric ribozyme was sufficiently distributed into human fibroblasts in vitro and rat gingivae in vivo. Chimeric ribozyme to TGF-beta1 significantly inhibited expression of TGF-beta1, type IV collagen, and fibronectin mRNAs and TGF-beta1 protein in human gingival fibroblasts. Mismatch ribozyme had no effect on expression of these molecules. Chimeric ribozyme to TGF-beta1 also significantly inhibited proliferation and DNA synthesis in gingival fibroblasts. Chimeric DNA-RNA ribozyme targeting TGF-beta1 may be a useful gene therapy agent for treatment of gingival hyperplasia.

Characterization and applications of CataCleave probe in real-time detection assays

Cycling probe technology (CPT), which utilizes a chimeric DNA–RNA–DNA probe and RNase H, is a rapid, isothermal probe amplification system for the detection of target DNA. Upon hybridization of the probe to its target DNA, RNase H cleaves the RNA portion of the DNA/RNA hybrid. Utilizing CPT, we designed a catalytically cleavable fluorescence probe (CataCleave probe) containing two internal fluorophores. Fluorescence intensity of the probe itself was weak due to Förster resonance energy transfer. Cleavage of the probe by RNase H in the presence of its target DNA caused enhancement of donor fluorescence, but this was not observed with nonspecific target DNA. Further, RNase H reactions with CataCleave probe exhibit a catalytic dose-dependent response to target DNA. This confirms the capability for the direct detection of specific target DNA through a signal amplification process. Moreover, CataCleave probe is also ideal for detecting DNA amplification processes, such as polymerase chain reaction (PCR) and isothermal rolling circle amplification (RCA). In fact, we observed signal enhancement proportional to the amount of RCA product formed. We were also able to monitor real-time PCR by measuring enhancement of donor fluorescence. Hence, CataCleave probe is useful for real-time monitoring of both isothermal and temperature-cycling nucleic acid amplification methods.

Chimeric DNA–RNA hammerhead ribozyme targeting transforming growth factor-β1 mRNA inhibits neointima formation in rat carotid artery after balloon injury

We designed and synthesized a chimeric DNA–RNA hammerhead ribozyme targeting transforming growth factor (TGF)-β1 mRNA and found that this ribozyme effectively and specifically inhibited growth of vascular smooth muscle cells. We examined the effects of the chimeric DNA–RNA hammerhead ribozyme targeting TGF-β1 mRNA on neointima formation and investigated the underlying mechanism to develop a possible gene therapy for coronary artery restenosis after percutaneous transluminal coronary angioplasty. Expression of mRNAs encoding TGF-β1, p27kip1, and connective tissue growth factor (CTGF) in carotid artery increased after balloon injury. Fluorescein-isothiocyanate (FITC)-labeled ribozyme was taken up into the midlayer smooth muscle of the injured carotid artery. Both 2 and 5 mg of ribozyme reduced neointima formation by 65% compared to that of controls. Ribozyme markedly decreased expression of TGF-β1 mRNA and protein in injured vessel. Mismatch ribozyme had no effect on expression of TGF-β1 mRNA protein in injured vessel. Ribozyme markedly decreased expression of fibronectin, p27kip1, and CTGF mRNAs in injured vessel, whereas a mismatch ribozyme had no effect on these mRNAs. These findings indicate that the chimeric DNA–RNA hammerhead ribozyme targeting TGF-β1 mRNA inhibits neointima formation in rat carotid artery after balloon injury with suppression of TGF-β1 and inhibition of extracellular matrix and CTGF. In conclusion, the hammerhead ribozyme against TGF-β1 may have promise as a therapy for coronary artery restenosis after percutaneous transluminal coronary angioplasty.

Chimeric DNA-RNA hammerhead ribozyme targeting PDGF A-chain mRNA specifically inhibits neointima formation in rat carotid artery after balloon injury.

Restenosis of the coronary artery after percutaneous transluminal coronary angioplasty (PTCA) occurs in 30-50% of patients and remains a major clinical problem. We developed ribozyme that targets platelet-derived growth factor (PDGF) A-chain mRNA as a gene therapy for restenosis after PTCA. Thus, we examined the effects of a chimeric DNA-RNA ribozyme targeting PDGF A-chain mRNA on neointima formation in rat carotid artery after balloon injury and evaluated its specificity for PDGF A-chain mRNA by microarray analysis. Rat carotid artery was injured with a 2F Fogarty catheter, and PDGF A-chain specific ribozyme was delivered to the injured artery with polyethylenimine. Two weeks after injury, the artery was removed, and the intima/media (I/M) ratio was evaluated. Six hours after injury, mRNA was extracted with oligo dT cellulose, and expression of PDGF A-chain mRNA was evaluated by reverse transcription-polymerase chain reaction. Expression of PDGF-AA protein was evaluated by Western blot analysis. Expression of 970 genes was evaluated by microarray (GeneChip, Affimetrix Inc). FITC-labeled ribozyme was taken up into the midlayer smooth muscle of the carotid artery until 24 h after balloon injury. Two and 5 microg of ribozyme significantly reduced neointima formation by 44 and 55% of control levels, respectively, in a dose-dependent manner. Ribozyme markedly inhibited expression of PDGF A-chain mRNA as well as production of PDGF-AA protein in injured vessels. Microarray analysis revealed that expression of 525 genes was increased after balloon injury. These genes included FLK-1, interleukin-1 receptor, retinoic acid receptor alpha2 isoform, heat shock protein, MAP kinase kinase, Fas antigen, G6Pase, PI-5-P-kinase, p38 MAP kinase, proliferating cell nuclear antigen, transforming growth factor-beta, extracellular signal-related kinase, and fibroblast growth factor receptor. With respect to expression of cytokine and growth factor mRNAs, the best ribozyme specifically inhibited expression of PDGF A-chain mRNA. Our chimeric DNA-RNA hammerhead ribozyme targeting PDGF A-chain mRNA inhibited neointima formation in rat carotid artery after balloon injury with specific inhibition of expression of PDGF A-chain mRNA, suggesting that this ribozyme may be useful for therapy of restenosis of coronary artery after PTCA.

Cleavage of a DNA–RNA–DNA/DNA chimeric substrate containing a single ribonucleotide at the DNA–RNA junction with prokaryotic RNases HII

We have analyzed the cleavage specificities of various prokaryotic Type 2 ribonucleases H (RNases H) on chimeric DNA–RNA–DNA/DNA substrates containing one to four ribonucleotides. RNases HII from Bacillus subtilis and Thermococcus kodakaraensis cleaved all of these substrates to produce a DNA segment with a 5′-monoribonucleotide. Consequently, these enzymes cleaved even the chimeric substrate containing a single ribonucleotide at the DNA–RNA junction (5′-side of the single ribonucleotide). In contrast, Escherichia coli RNase HI and B. subtilis RNase HIII did not cleave the chimeric substrate containing a single ribonucleotide. These results suggest that bacterial and archaeal RNases HII are involved in excision of a single ribonucleotide misincorporated into DNA.

Intracellular inhibition of hepatitis B virus S gene expression by chimeric DNA-RNA phosphorothioate minimized ribozyme.

Chronic hepatitis B virus (HBV) infection is a major problem in Asia. Current therapies for chronic hepatitis B have limited efficacy. The successful use of ribozymes for intracellular inhibition of HBV gene expression was recently reported. As an alternative to ribozymes, the use of DNA-containing, phosphorothioate-modified, minimized hammerhead ribozymes (minizymes) to inhibit hepatitis B surface antigen (HBsAg) expression and viral replication was investigated. Such molecules can be synthesized and supplied exogenously. Two conserved sites within the HBsAg open reading frame (ORF) were targeted. PLC/PRF5 cells or 2.2.15 cells were treated with minizymes or antisense oligomers to assess the effects on cell viability, HBsAg expression, and viral DNA production. Treatment with the minizyme, MZPS1, resulted in >80% inhibition of HBsAg expression in PLC/PRF5 cells. MZPS1 had more inhibitory effect than the antisense oligonucletoide target at the same region, whereas the control minizyme had little effect. Another gene-specific minizyme, MZPS2, did not show any effect. Treated cells remained fully viable. Treatment of 2.2.15 cells with MZPS1 also led to decreased HBsAg expression. In addition, a 2.3-fold decrease in viral production was observed. Our data showed that minizymes can inhibit HBV gene expression and may potentially be useful for clinical therapy against chronic HBV infection.

Ribozyme-mediated inhibition of rat leukocyte-type 12-lipoxygenase prevents intimal hyperplasia in balloon-injured rat carotid arteries.

12-Lipoxygenase (12-LO) products of arachidonate metabolism have growth and chemotactic effects in vascular smooth muscle cells. We have also recently demonstrated increased 12-LO mRNA and protein expression in the neointima of balloon-injured rat carotid arteries. In this study, we evaluated whether 12-LO activation plays a role in neointimal thickening in this rat model by using a specific ribozyme (Rz) directed to rat 12-LO. We designed a chimeric DNA-RNA hammerhead Rz to cleave rat leukocyte-type 12-LO mRNA. This Rz dose-dependently cleaved a 166-nucleotide target 12-LO mRNA substrate in vitro and reduced 12-LO mRNA and protein expression in rat vascular smooth muscle cells. A control mutant Rz (MRz) with a point mutation in the catalytic site was inactive. To test the in vivo efficacy of the 12-LO Rz, the left common carotid arteries of rats were injured with a balloon catheter. The distal half of the injured arteries was treated with Rz or MRz mixed with lipofectin. The proximal half received only lipofectin. Twelve days after injury, intima-to-media ratios were significantly lower in the Rz-treated sections than in untreated sections from the same rat (0.742+/-0.16 versus 1.749+/-0.12, P:<0.001). In contrast, the MRz had no significant effect. These results indicate the important role of the leukocyte-type 12-LO pathway in restenosis in response to injury.