Determination of the Role of the Human RNase H1 in the Pharmacology of DNA-like Antisense Drugs

Although ribonuclease H activity has long been implicated as a molecular mechanism by which DNA-like oligonucleotides induce degradation of target RNAs, definitive proof that one or more RNase H is responsible is lacking. To date, two RNase H enzymes (H1 and H2) have been cloned and shown to be expressed in human cells and tissues. To determine the role of RNase H1 in the mechanism of action of DNA-like antisense drugs, we varied the levels of the enzyme in human cells and mouse liver and determined the correlation of those levels with the effects of a number of DNA-like antisense drugs. Our results demonstrate that in human cells RNase H1 is responsible for most of the activity of DNA-like antisense drugs. Further, we show that there are several additional previously undescribed RNases H in human cells that may participate in the effects of DNA-like antisense oligonucleotides. Although ribonuclease H activity has long been implicated as a molecular mechanism by which DNA-like oligonucleotides induce degradation of target RNAs, definitive proof that one or more RNase H is responsible is lacking. To date, two RNase H enzymes (H1 and H2) have been cloned and shown to be expressed in human cells and tissues. To determine the role of RNase H1 in the mechanism of action of DNA-like antisense drugs, we varied the levels of the enzyme in human cells and mouse liver and determined the correlation of those levels with the effects of a number of DNA-like antisense drugs. Our results demonstrate that in human cells RNase H1 is responsible for most of the activity of DNA-like antisense drugs. Further, we show that there are several additional previously undescribed RNases H in human cells that may participate in the effects of DNA-like antisense oligonucleotides. RNase H hydrolyzes RNA in RNA-DNA hybrids (1Stein H. Hausen P. Science. 1969; 166: 393-395Crossref PubMed Scopus (179) Google Scholar). RNase H activity appears to be ubiquitous in eukaryotes and bacteria (2Itaya M. Kondo K. Nucleic Acids Res. 1991; 19: 4443-4449Crossref PubMed Scopus (63) Google Scholar, 3Itaya M. McKelvin D. Chatterjie S.K. Crouch R.J. Mol. Gen. Genet. 1991; 227: 438-445Crossref PubMed Scopus (54) Google Scholar, 4Kanaya S. Itaya M. J. Biol. Chem. 1992; 267: 10184-10192Abstract Full Text PDF PubMed Google Scholar, 5Busen W. J. Biol. Chem. 1980; 255: 9434-9443Abstract Full Text PDF PubMed Google Scholar, 6Rong Y.W. Carl P.L. Biochemistry. 1990; 29: 383-389Crossref PubMed Scopus (34) Google Scholar, 7Eder P.S. Walder J.A. Biochimie (Paris). 1993; 75: 6472-6479Crossref Scopus (103) Google Scholar). Although RNases H constitutes a family of proteins of varying molecular weight, the nucleolytic activity and substrate requirements appear to be similar for the various isotypes. For example, all RNases H studied to date function as endonucleases exhibiting limited sequence specificity and requiring divalent cations (e.g. Mg2+, Mn2+) to produce cleavage products with 5′ phosphate and 3′ hydroxyl termini (8Crouch R.J. Dirksen M.L. Linn S.M. Roberts R.J. Cold Spring Harbor. Cold Spring Harbor Press, Plainview, New York1982: 211-254Google Scholar). Although a number of viral and bacterial polymerases and exonucleases have been shown to have RNase H activities (8Crouch R.J. Dirksen M.L. Linn S.M. Roberts R.J. Cold Spring Harbor. Cold Spring Harbor Press, Plainview, New York1982: 211-254Google Scholar), in mammalian cells only two classes of RNase H enzymes have been identified (5Busen W. J. Biol. Chem. 1980; 255: 9434-9443Abstract Full Text PDF PubMed Google Scholar, 9Eder P.S. Walder J.A. J. Biol. Chem. 1991; 266: 6472-6479Abstract Full Text PDF PubMed Google Scholar, 10Frank P. Albert S. Cazenave C. Toulme J.J. Nucleic Acids Res. 1994; 22: 5247-5254Crossref PubMed Scopus (40) Google Scholar). These enzymes were shown to differ with respect to co-factor requirements and were shown to be inhibited by sulfhydryl reagents (10Frank P. Albert S. Cazenave C. Toulme J.J. Nucleic Acids Res. 1994; 22: 5247-5254Crossref PubMed Scopus (40) Google Scholar, 11Wu H. Lima W.F. Crooke S.T. Antisense Nucleic Acid Drug Dev. 1998; 8: 53-61Crossref PubMed Scopus (54) Google Scholar). Although the biological roles of the mammalian enzymes are not fully understood, it has been suggested that mammalian RNase H1 may be involved in replication and that the RNase H2 enzyme may be involved in transcription (12Busen W. Peters J.H. Hausen P. Eur. J. Biochem. 1977; 74: 203-208Crossref PubMed Scopus (48) Google Scholar, 13Turchi J.J. Huang L. Murante R.S. Kim Y. Bambara R.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9803-9807Crossref PubMed Scopus (168) Google Scholar). Recently, an RNase H1 knock-out in the mouse was shown to be embryonically lethal, and it showed that RNase H1 is involved in mitochondrial DNA replication (14Cerritelli S.M. Frolova E.G. Feng C. Grinberg A. Love P.E. Crouch R.J. Mol. Cell. 2003; 11: 807-815Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Two human RNase H genes have been cloned and expressed (11Wu H. Lima W.F. Crooke S.T. Antisense Nucleic Acid Drug Dev. 1998; 8: 53-61Crossref PubMed Scopus (54) Google Scholar, 15Frank P. Braunshofer-Reiter C. Wintersberger U. Grimm R. Busen W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12872-12877Crossref PubMed Scopus (68) Google Scholar, 16Frank P. Braunshofer-Reiter C. Poeltl A. Holzmann K. Biol. Chem. 1998; 379: 1407-1412Crossref PubMed Scopus (23) Google Scholar, 17Cerritelli S.M. Crouch R.J. Genomics. 1998; 53: 300-307Crossref PubMed Scopus (47) Google Scholar). RNase H1 is a 286-amino acid protein and is expressed ubiquitously in human cells and tissues (11Wu H. Lima W.F. Crooke S.T. Antisense Nucleic Acid Drug Dev. 1998; 8: 53-61Crossref PubMed Scopus (54) Google Scholar). The amino acid sequence of human RNase H1 displays strong homology with RNase H1 from yeast, chicken, Escherichia coli, and mouse (11Wu H. Lima W.F. Crooke S.T. Antisense Nucleic Acid Drug Dev. 1998; 8: 53-61Crossref PubMed Scopus (54) Google Scholar). The human RNase H2 enzyme is a 299-amino acid protein with a calculated mass of 33.4 kDa and has also been shown to be ubiquitously expressed in human cells and tissues (15Frank P. Braunshofer-Reiter C. Wintersberger U. Grimm R. Busen W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12872-12877Crossref PubMed Scopus (68) Google Scholar). Human RNase H2 shares strong amino acid sequence homology with RNase H2 from Caenorhabditis elegans, yeast, and E. coli (15Frank P. Braunshofer-Reiter C. Wintersberger U. Grimm R. Busen W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12872-12877Crossref PubMed Scopus (68) Google Scholar, 18Wu H. Lima W.F. Crooke S.T. J. Biol. Chem. 1999; 274: 28270-28278Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). The properties of the cloned and expressed human RNase H1 have recently been characterized; many of the properties observed for human RNase H1 are consistent with the E. coli RNase H1 isotype, (e.g. the cofactor requirements, substrate specificity, and binding specificity) (18Wu H. Lima W.F. Crooke S.T. J. Biol. Chem. 1999; 274: 28270-28278Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 19Lima W.F. Crooke S.T. Biochemistry. 1997; 36: 390-398Crossref PubMed Scopus (111) Google Scholar). In fact, the carboxyl-terminal portion of human RNase H1 is highly conserved with the amino acid sequence of the E. coli enzyme. The glutamic acid and two aspartic acid residues of the catalytic site, as well as the histidine and aspartic acid residues of the proposed second divalent cation-binding site of the E. coli enzyme, are conserved in human RNase H1 (20Kanaya S. Katsuda-Nakai C. Ikebara M. J. Biol. Chem. 1991; 266: 11621-11627Abstract Full Text PDF PubMed Google Scholar, 21Nakamura H. Oda Y. Iwai S. Inoue H. Ohtsuka E. Kanaya S. Kimura S. Katsuda C. Katayanagi K. Morikawa K. Miyashiro H. Ikehara M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11535-11539Crossref PubMed Scopus (192) Google Scholar, 22Katayanagi K. Miyagawa M. Matsushima M. Ishikawa M. Kanaya S. Ikehara M. Matsuzaki T. Morikawa K. Nature. 1990; 347: 306-309Crossref PubMed Scopus (307) Google Scholar, 23Yang W. Hendrickson W.A. Crouch R.J. Satow Y. Science. 1990; 249: 1398-1405Crossref PubMed Scopus (451) Google Scholar). In addition, the lysine residues within the highly basic α-helical substrate-binding region of E. coli RNase H1 are also conserved in the human enzyme. Site-directed mutagenesis of the catalytic amino acids and the basic residues of the substrate-binding domain of human RNase H1 showed that these conserved residues are required for activity (24Landt O. Grunert H. Hanh U. Gene. 1990; 96: 125-128Crossref PubMed Scopus (638) Google Scholar). Recently a novel redox-dependent regulator element has been reported for the enzyme (25Lima W. Wu H. Nichols J.G. Manalili S.M. Drader J.J. Hofstadler S.A. Crooke S.T. J. Biol. Chem. 2003; 278: 14906-14912Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). In contrast to human RNase H1, nothing is known about the detailed enzymological properties of human RNase H2 because the cloned and expressed enzyme is inactive. Antisense oligonucleotides (ASOs) 1The abbreviations used are: ASO, antisense oligonucleotide; siRNA, small interference RNA; pfu, plaque-forming unit. 1The abbreviations used are: ASO, antisense oligonucleotide; siRNA, small interference RNA; pfu, plaque-forming unit. have proven of value in determining gene functions and may be of value as a new therapeutic class (26Crooke S.T. Abraham D.J. Burger's Medicinal Chemistry. 6th Ed. Vol. 5. John Wiley and Sons, Inc., New York2003: 115-166Google Scholar). Once ASOs bind via Watson-Crick hybridization to target RNAs, they may work through a variety of mechanisms of action (26Crooke S.T. Abraham D.J. Burger's Medicinal Chemistry. 6th Ed. Vol. 5. John Wiley and Sons, Inc., New York2003: 115-166Google Scholar, 27Crooke S.T. Biochim. Biophys. Acta. 1999; 1489: 30-42Google Scholar, 28Crooke S.T. Crooke S.T. Antisense Technology: Principles, Strategies, and Applications. Marcel Dekker, Inc., New York2001: 1-28Google Scholar). DNA-like ASOs are thought to work by creating a substrate for cellular RNases H after binding to target RNAs (28Crooke S.T. Crooke S.T. Antisense Technology: Principles, Strategies, and Applications. Marcel Dekker, Inc., New York2001: 1-28Google Scholar). Although a large body of inferential evidence supports this concept, direct proof of the mechanism is lacking, and to date compelling evidence has not been reported with regard to which of the RNases H may be responsible (27Crooke S.T. Biochim. Biophys. Acta. 1999; 1489: 30-42Google Scholar, 28Crooke S.T. Crooke S.T. Antisense Technology: Principles, Strategies, and Applications. Marcel Dekker, Inc., New York2001: 1-28Google Scholar, 29Giles R.V. Spiller D.G. Tidd D.M. Antisense Res. Dev. 1995; 5: 23-31Crossref PubMed Scopus (102) Google Scholar). The purpose of this study is to unequivocally demonstrate that DNA-like ASOs induce reduction of target RNAs by creating RNA-ASO duplexes that serve as substrates for mammalian cellular RNases H and to determine which of the two known mammalian RNases H plays a significant role in this process. To achieve these objectives, we have altered the level of expression and cellular activity of the RNase H1 and then determined the effects of these alterations on the ability of DNA-like ASOs to reduce various RNA targets. Because RNase H1 knock-outs have been reported to be lethal, we have employed overexpression and antisense or double-stranded siRNA (siRNA) techniques to alter levels of RNase H1. Our studies demonstrate that human RNase H1 is critically involved in the effects of DNA-like ASOs. Further, our data suggest that there may be other RNases H in mammalian cells that contribute to the activity of DNA-like ASOs. Oligonucleotide Synthesis—Synthesis and purification of chimeric 2′-O-methoxyethyl (MOE)/deoxy phosphorothioate-modified oligonucleotides were as previously described (30McKay R. Miraglia L. Cummins L. Owens S. Sasmor H. Dean N.M. J. Biol. Chem. 1999; 274: 1715-1722Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 31Baker B.F. Lot S.S. Condon T.P. Cheng-Flournoy S. Lesnik E.A. Sasmor H.M. Bennett C.F. J. Biol. Chem. 1997; 272: 11994-12000Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). Sequences of oligonucleotides and placement of 2′-O-MOE modifications are detailed below. Unmodified oligodeoxynucleotides were purchased from Invitrogen. The oligoribonucleotides were purchased from Dharmacon Research, Inc. (Boulder, CO). siRNA duplexes were formed in the solution containing 20 μm each oligoribonucleotide, 100 mm potassium acetate, 30 mm HEPES-KOH, pH 7.4, 2 mm were for and for The of the ASOs used in the study are residues are are and RNase The siRNA sequence siRNA RNase The ASO, is a of each The was by of and each of the oligonucleotides. The were employed and were used The of each in the was to The siRNA in are the RNA or other RNA duplexes that were to the human RNase H1 acids to the and acids to the of the protein number were to with and used to in E. D. a Cold Spring Harbor Cold Spring New Scholar). were also the human RNase H1 acid H. Lima W.F. Crooke S.T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). were with protein of H1 were then on by the to a for the of protein or overexpression of human RNases H1, the RNase H1 region was by and cloned the The were by DNA and then the was by the of the of The was also by the shares all with the RNase for the The were by or purification viral and and cells were in with in or and were in with and cells were in with and and all were purchased from Invitrogen. For of or siRNA, cells were with a of or siRNA in the was from the cells and with containing and the cells or second For of were the and or from cells or mouse liver were as described Nucleic Acids Res. 11: PubMed Scopus Google protein were by the of The were in and then by mass were used to determine the protein The proteins were to a and for human RNases H The were the and RNA was from human of RNA were on a to and to the a was by human RNase H1, or DNA in for 2 were in a of for 30 were RNA the RNA was from an and a to the The RNA was with the RNA expression was as described J. C. S. Biochem. 1999; PubMed Scopus Google Scholar). RNA was in a of containing mm of each mm 2 of and of ribonuclease transcription was for 30 by of 30 and an sequence The were Human number and Human number and Human protein number and Human RNase H1 number and protein number and were purchased from were with various of in or with in by with the RNase or viral in of or the or viral in of by to the RNA was from mouse liver RNase were to the and a were used as 20 of RNA was on were then on a expression levels were to or levels in each and as the of determine the in many the were with a in the and the To the of of each on each and were For in the RNase the value with was of Human RNase overexpression of human RNase H1, a of containing the shown in was that the enzyme was in and were observed after In addition, the enzyme be in and cells not a of the from the that the human RNase H1 enzyme was and with the enzyme from To determine the RNase H1 was we employed the previously human RNase H1 be and was in this Human RNase H1 activity was in the and of To that the activity was human RNase H1, the enzyme was from cells and then to the that the enzyme was in the and of the human RNase H1 in activity in the of Human RNase H1 the of DNA-like the of overexpression of the RNases H on the of DNA-like and cells were with the or RNase H1 containing and then the effects of several well ASOs Drug 1997; Google Scholar, R.S. J. T. L. C. M. Drug 1997; Google Scholar, R. Dean D. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, R. M. O. Dean D. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar, M. W.A. D. Dean N.M. S. PubMed Scopus Google on the of target RNAs that the of ASOs to bind to human or 2 were by overexpression of human RNase H1 in that similar results were observed in that similar results were observed or were The for each each are shown the Because DNA-like ASOs are used in and are in in (26Crooke S.T. Abraham D.J. Burger's Medicinal Chemistry. 6th Ed. Vol. 5. John Wiley and Sons, Inc., New York2003: 115-166Google Scholar), we our to in To this we that human RNase H1 be in mouse cells was observed in human overexpression of human RNase H1 the of a DNA-like to bind to mouse RNA To determine overexpression of human RNase H1 in mouse liver the of DNA-like we the effects of a well mouse J. J. R. K. K. Dean N.M. PubMed Scopus Google Scholar). of were with the and human RNase as described that human RNase H1 was in the liver of the that were with the containing the The human RNase H1 expressed in mouse liver was in the the of overexpression was that the the reduction of RNA in mouse To the effects of overexpression of RNase H1 on the of the ASO, the were and to data were then used to the shown in The effects of overexpression of human RNase H1 were by of protein with a not the there was evidence of significant liver and the the and human RNase H1 not the of in the liver of with of were similar in with or RNase 20 RNase H1, 30 RNase H1, RNase H1, of the effects observed were to to in of the liver as a of of Human RNase H1 the of DNA-like the overexpression we have RNase H1 levels and the effects of enzyme levels on the of DNA-like ASOs. DNA-like ASOs and siRNA were identified for enzymes by cellular as previously described (26Crooke S.T. Abraham D.J. Burger's Medicinal Chemistry. 6th Ed. Vol. 5. John Wiley and Sons, Inc., New York2003: 115-166Google Scholar, S. Bennett C.F. Crooke S.T. Dean N.M. B.F. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). To the most ASOs and to human RNase H1, we to bind to in the The most in human RNase H1 was in the region of the RNA number The most siRNA for RNase H1 was in the region of the The effects of various of each of the were then the and siRNA in of RNase H1 RNA in and cells the and the thought to be human RNase H1 RNA were Further, the RNase H1 activity in was as shown in the were effects on RNase H2 levels not The of for the and was not that reduction in the levels of human RNase H1 the of the RNA in of the siRNA to human RNase H1 in a reduction in the of the Further, there is a correlation the reduction of RNA by the and the cellular level of human RNase H1 or and that an of the for the not demonstrate antisense activity there was human RNase H1 that an human RNase H1 the of the in and These results were with the effects of to RNase H1. RNase in Human Two are by the RNase H1 RNase H1 is required for the activity of DNA-like ASO, was the cellular RNase H1 was by more only RNase H1 is involved in the activities of DNA-like the RNase H1 that not to activity RNase observed several additional molecular mass as well as several molecular mass from in the These were observed in a variety of cells the was mm The level of activity and the number of varied from to and from to the was in the of mm the RNase H activity were more and one molecular mass activity was observed in all studied and that reduction of RNase H1 with an or siRNA in and cells the RNase H1 of activity and on the molecular mass of RNase H In we of RNase H1 in The after of RNase H1 RNase H1. Further, in the there was RNase H1 activity in the in the several of the novel RNase H activity The results are from similar with not These results show that there are several previously RNase H activities in human cells that are not RNase H1 or H2 are in a the RNA level with ASOs or with RNase H1 or H2 the level of activities of the RNases H. Although it has been that DNA-like ASOs target RNA reduction by binding to the target RNA and creating a that as a substrate for RNase definitive proof that this mechanism is responsible for the observed effects in mammalian cells and is (27Crooke S.T. Biochim. Biophys. Acta. 1999; 1489: 30-42Google Scholar, 28Crooke S.T. Crooke S.T. Antisense Technology: Principles, Strategies, and Applications. Marcel Dekker, Inc., New York2001: 1-28Google Scholar). In the of E. coli RNase H or human RNase H1 to duplexes results in degradation of the target RNA (18Wu H. Lima W.F. Crooke S.T. J. Biol. Chem. 1999; 274: 28270-28278Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, S.T. K. L. R. Lesnik E.A. Biochem. J. 1995; PubMed Scopus Google Scholar, W.F. Crooke S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). The ability of DNA-like ASOs to a reduction in target RNAs in cells has been many as well (28Crooke S.T. Crooke S.T. Antisense Technology: Principles, Strategies, and Applications. Marcel Dekker, Inc., New York2001: 1-28Google Scholar). in the of a DNA-like that in of the ability of the to serve as a substrate for RNase H in have been reported to in a of target RNA reduction in cells with the (28Crooke S.T. Crooke S.T. Antisense Technology: Principles, Strategies, and Applications. Marcel Dekker, Inc., New York2001: 1-28Google Scholar, H. S.M. Lima W.F. Bennett C.F. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar). R.V. Spiller D.G. Tidd D.M. Antisense Res. Dev. 1995; 5: 23-31Crossref PubMed Scopus (102) Google used to cleavage products from in cells with a DNA-like of these studies that DNA-like ASOs reduce target RNA by RNase H has the RNase H that is responsible been Because knock-outs of human RNase H1 are (5Busen W. J. Biol. Chem. 1980; 255: 9434-9443Abstract Full Text PDF PubMed Google Scholar, S.M. Frolova E.G. Feng C. Grinberg A. Love P.E. Crouch R.J. Mol. Cell. 2003; 11: 807-815Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar), we have to human RNase H1 and to and siRNA reduction of RNase H1 as to determine RNase H is required for target RNA reduction by DNA-like ASOs. DNA-like ASOs RNA by as for Human RNase the in this we direct evidence that DNA-like ASOs work via an RNase H show that overexpression of human RNase H1 the of several ASOs several target RNAs in several human also demonstrate that overexpression of human RNase H1 in mouse cells and mouse liver the of DNA-like ASOs and reduction of RNase H1 results in a of for to RNA target in several human and overexpression and reduction of RNase H1 demonstrate that RNase H1 is involved in the effects of DNA-like ASOs. is the direct of the role of an RNase H in the activity of a DNA-like The differ from a M. M. Eur. J. Biochem. PubMed Scopus (40) Google that RNase H2 plays an role in the activity of DNA-like ASOs on In they that RNase H2 be involved because it is more and in from RNase H1. direct evidence on of the levels of enzymes a more compelling RNases H a in the of DNA-like of siRNA or of RNase H1 to activity that RNase H1 plays a role other RNases H may be RNase H1 is have identified several RNases H that are not by to RNase H1 or H2 and These enzymes are not by ASOs or to RNase H1. are in the that they are as and be of are more in the of Mg2+, and activities are from to and within These data suggest that these enzymes differ from the cloned mammalian RNase H and that they are from RNase H1, because the levels of these enzymes the novel activities we have may be chimeric proteins with or other activities to a with RNase H are in the of and the for Antisense that human RNase H1 plays a role in the activities of DNA-like ASOs that additional studies that the substrate and for RNase H1 of antisense The that in RNase H1 activity with in that of RNase H1 to the cleavage of the RNA by the enzyme is for that these Medicinal that the of chimeric the placement of DNA-like to of or of the enzyme are in also be to the novel RNases H and to ASOs for and for Bennett and for and for