MXPA01002953A - Hairpin hybridizer molecules for modulation of gene expression - Google Patents

Abstract

The invention features novel hairpin hybridizer nucleic acid molecules which are able to modulate gene expression;useful in target validation, gene function identification and in human therapeutics.

Description

HORRIBLE HIBRIDING MOLECULES TO MODULATE THE EXPRESSION OF GENES RELATED REQUESTS This patent application claims the benefit of Hartmann et al., USSN 60/101, 174, filed on September 21, 1998 entitled "HAIRPIN HYBRIDIZER MOLECULES FOR MODULATION OF GENE EXPRESSION". Therefore, this application is incorporated in the present invention for reference in its entirety including the drawings.

BACKGROUND OF THE INVENTION This invention relates to nucleic acid molecules, which the Applicant calls "fork hybridizing" (HPH) molecules which can modulate the expression of genes by hybridization to the target RNA with an improved specificity thereby blocking the translation of such RNA objective. What follows is a discussion of the relevant technique, none of which is considered a prior art to the present invention. Since the discovery of the underlying mechanisms of gene expression, specifically transcription and translation based on nucleic acid, a great deal of effort has been made to block or altering these processes with a variety of purposes, such as understanding biology, gene function, disease processes, and identifying novel therapeutic goals. Methods that involve the use of nucleic acid molecules to modulate gene expression have gained popularity in recent years. For example, nucleic acid molecules that can bind to specific mRNA sequences have been designed through the interaction of Watson-Cric base pairing and translation block (Crooke, 1996, Medicinal Res. Rev. 16, 319-344 ). Another method involves the formation of DNA complexes with oligonucleotides that form triplex complexes to prevent transcription of the ligated DNA sequences thereby inhibiting gene expression (Kim et al., 1998, Biochemistry, 37, 2299-2304). The interaction of antisense oligonucleotides, 2-5A antisense chimeric molecules, or ribozymes with target RNA molecules has been used to modulate gene expression. All of these nucleic acid molecules are highly specific with their target mating sequences and therefore may offer less toxicity compared to traditional methods, such as chemotherapy. The concept of inhibition of gene expression through an antisense mechanism is derived in part from the mechanisms found in nature. It has been found that prokaryotic systems use complementary RNA molecules to inhibit translation (Lacetena et al., 1983, Blood 170, 635-650). Simons et al., 1983, Cell 34, 673-682; Mizuno eí al., 1984, Proc. Natl. Acad Sci. (E.U.A.) 81, 1966-1970). For example, expression of the OmpF and OmpC genes of E. coli (outer membrane proteins) is regulated by an antisense RNA mechanism (Mukopadhyay &Roth, 1996, Critical Rev. In Oncogenesis 7, 151-190). Antisense oligonucleotides can be used to negatively regulate the target mRNA by a number of different mechanisms. The specific character of these reagents is determined by the primary sequence (GC content, sequence length), chemistry (ribonucleotides, deoxyribonucleotides, chemically modified nucleotides) of the antisense molecule and the presence of pseudo-target sequences. The pseudo-targets are nucleic acid sequences, which may have identity or sequence homology with an objective sequence. The number of pseudo-targets for a given sequence, especially human genes, is mostly unknown up to this point, since only a small fraction of the human genome has been sequenced at present. Lizardi eí al., Patent of E.U.A. No. 5, 312,728, describe a self-inhibiting nucleic acid molecule, known as a molecular switch, used for the detection of target nucleic acid molecules. The molecular switch consists of a probe sequence of 20 to 60 nucleotides that can hybridize to an objective sequence and 5 'and 3' sequences of at least 10 nucleotides in length that can hybridize to one another intramolecularly.

Engdahl et al., 1997, Nucleic Acids Research 25, 3218-3227, describe the use of an RNA cassette system to silence the lacl gene. The molecules used consisted of a hairpin structure, which was used for recognition of the target sequence and an inhibitor region which was either an antisense sequence or a ribozyme sequence. Delihas et al., 1997, Nature Biotech 15, 751-753, describe the formation of non-canonical base pairs using natural antisense RNA and target RNA. Stinchcomb et al., PCT International Publication No. WO 95/23225, describe an RNA molecule with an intramolecular stem-locus structure greater than or equal to 8 base pairs.

BRIEF DESCRIPTION OF THE INVENTION This invention relates to nucleic acid molecules that can bind and block the function of target nucleic acid molecules, thereby modulating cellular or viral mechanisms including splicing, editing, gene expression or replication, and translation. Specifically, the invention relates to novel nucleic acid molecules with a secondary hairpin structure that can negatively regulate the expression of proteins by binding (steric blocker) and optionally facilitates the cutting of target RNA through an RNAse H or other mechanism. For simplicity and ease of understanding of this invention, the nucleic acid molecules of the present invention should be preferred as hairpin hybridizing molecules (HPH). In particular, the applicant describes the use of these HPH molecules to negatively regulate gene expression in bacterial, microbiological, fungal, eukaryotic systems including plants, or mammalian cells. Negative regulation of specific target sequences could have either a therapeutic effect in many diseases or disease states or help in the identification of gene function and / or novel therapeutic therapeutic targets. The HPH molecules of the present invention can be used for in vitro or in vivo applications well known in the art. The present invention features a method for modulating the function of an objective sequence in a cell using HPH molecules. HPH molecules include a target binding region and a hairpin region, in which the target binding region can be linked to the target sequence in a form that is sequence specific in vitro or in vivo to modulate the function of the objective sequence. The hairpin region of the HPH molecules provides an improved specificity characteristic to the HPH molecule. The hairpin region is expected to provide improved resistance to nuclease degradation, it is expected to assist the HPH molecule with the location within a cell, and it is expected to assist in an improved absorption of HPH molecules by cells compared to a molecule lacking such a hairpin structure. The target binding region of the HPH molecule could also include an activation region of RNase H in which such a region includes a nucleotide sequence greater than or equal to 4 deoxyribonucleotides with intemucleotide bonds of the phosphorothioate, phosphodiester, phosphorodithioate, arabin, type. '-fluoroarabino and / or 5'-thiophosphate. The RNAse H activation region interacts with the target RNA to form a DNA: RNA complex that is recognized by the cellular RNAse H enzyme, which binds to the DNA: RNA complex and cuts the RNA portion of the DNA: RNA complex. Such a cut of the target RNA mediated by RNase H causes the target RNA to lose its normal function causing the inhibition of its translation into proteins, its replication, its packaging into viral particles, or other functions. Therefore in a first aspect, a method for modulating the function, such as expression, of an objective sequence, preferably in a cell, comprises the step of contacting said target sequence with an HPH nucleic acid molecule under conditions suitable for modulating the expression of said target sequence, in which the HPH nucleic acid molecule includes the following formulas: FORMULA I in which, each P, Y, N and M independently represent a nucleotide that could be the same or different; • indicates the formation of hydrogen bonds between two adjacent nucleotides; N 'is a complementary nucleotide for N; or is an integer greater than or equal to 3, more specifically 4, 5, 6, 7, 8 or 9; k is zero or is an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20; t is zero or is an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20; w is an integer greater than or equal to 4 and preferably less than about 100, more specifically 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; (P) t and (P) k are independently oligonucleotides, which preferably include at least one position other than deoxynucleotide (e.g. nucleotide containing 2'-H); (P) and (P) k could include linkers of the phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate and the like type or a combination thereof; k and t could have the same length (k = t) or they could have different lengths (k? t); (M) w is a sequence of oligonucleotides whose inter-nucleotide linkers include linkers of the phosphodiester, phosphorothioate, 5'-thiophosphate, or methylphosphonate type or a combination thereof; t, k and w could have the same length (k = t = w), or they could have different lengths (k? t? w) or (k = t? w) or (k? t = w) or (k = w? t); at least one or more of (P) t and (P) k, and (M) w is an oligonucleotide of sufficient length to interact independently stably with a target sequence (the target could be an RNA, a DNA or mixed RNA / DNA polymers); r and f are independently an integer greater than or equal to zero, specifically 1, 2, 3, 4, 5, 10, or 15; each B and B 'independently represent a blocking structure, which independently could be present or absent; when r = 0, and f = 0, B and / or B ', when present, they are attached to (N »N') 0; and represents a chemical bond (for example a phosphate ester bond, an amide bond, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate or others known in the art).

FORMULA II in which, each P, N and M independently represent a nucleotide that could be the same or different; • indicates the formation of hydrogen bonds between two adjacent nucleotides; N 'is a complementary nucleotide for N; or is an integer greater than or equal to 3, more specifically 4, 5, 6, 7, 8 or 9; k is zero or is an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20; t is zero or is a whole number greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20; k1 is zero or is an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20; t1 is zero or is an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20; w is an integer greater than or equal to 4 and preferably less than about 100, more specifically 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; (P) t and (P) k, (P) H and (P) M are independently oligonucleotides, which preferably include at least one position other than deoxynucleotide (e.g. nucleotide containing 2'-H); (P) t and (P) ?, (P) t? and (P) M could include linkers of the phosphodiester, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate and the like type or a combination thereof; k and t could have the same length (k = t) or they could have different lengths (k? t); k1 and t1 could have the same length (k1 = t1) or they could have different lengths (k1? t1); (M) w is a sequence of oligonucleotides whose inter-nucleotide linkers include linkers of the phosphodiester, phosphorothioate, 5'-thiophosphate, methylphosphonate, or phosphorodithioate type or a combination thereof; t, k and w could have the same length (k = t = w) or they could have different lengths (k? t? w) or (k = t? w) or (k? t = w) or (k = w? t ); t1, k1 and w could have the same length (k1 = t1 = w) or could have different lengths (k1? t1? w) or (k1 = t1? w) or (k1? t1 = w) or (k1 = w? t1); at least one or more of (P) t, (P) k, (P) t ?, (P) ki and (M) w is a oiigonucleotide of sufficient length to independently interact stably with a target sequence (the target could be an RNA, DNA or mixed RNA / DNA polymers); and represents a chemical bond (for example a phosphate ester linkage, an amide bond, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate or others known in the art).

FORMULA lll in which, each P, N and M independently represent a nucleotide that could be the same or different; • indicates the formation of hydrogen bonds between two adjacent nucleotides; N 'is a complementary nucleotide for N; or is an integer greater than or equal to 3, more specifically 4, 5, 6, 7, 8 or 9; k is zero or is an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20; t is zero or is an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20; w is an integer greater than or equal to 4 and preferably less than about 100, more specifically 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; (P) t and (P) k are oligonucleotides, which preferably include at least one position other than deoxynucleotide (e.g., nucleotide containing 2'-H); each of (P) t and (P) k could include linkers of the phosphodiester, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methyphosphonate and the like type or a combination thereof; k and t could have the same length (k = t) or they could have different lengths (k? t); (M) w is a sequence of oligonucleotides whose inter-nucleotide linkers could include linkers of the phosphodiester, phosphorothioate, 5'-thiophosphate, methylphosphonate or phosphorodithioate type, or a combination thereof; t, k and w could have the same length (k = t = w), or they could have different lengths (k? t? w) or (k = t? w) or (k? t = w) or (k = w? t); at least one or more of each said (P) ty (P) k, and (M) w is an oligonucleotide of sufficient length to independently interact stably with a target sequence (the target could be an RNA , a DNA or mixed RNA / DNA polymers); D and E are oligonucleotides, which are greater than or equal to 4 and preferably less than 100 nucleotides in length, specifically 6, 7, 8, 9, 10, 11, 12, 15, 20 or 30 and have length sufficient for it to independently interact stably with a target sequence (the target could be an RNA, a DNA or mixed RNA / DNA polymers). The oligonucleotides D and E can be symmetric (D = E in length) or can be asymmetric (D? E in length); each B and B 'independently represent a blocking structure, which independently could be present or absent; and represents a chemical bond (for example a phosphate ester bond, an amide bond, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate or others known in the art).

FORMULA IV wherein, N represents a ribonucleotide which could be the same or different; N 'is a complementary nucleotide for N; • indicates the formation of hydrogen bonds between two adjacent ribonucleotides; or is an integer greater than or equal to 3 and less than or equal to 9, more specifically 4, 5, 6, 7, 8 or 9; S, A and B are oligoribonucleotides which are independently equal to 5 and preferably less than 100 nucleotides in length, more specifically 6, 7, 8, 9, 10, 11, 12, 15, 20, or 30; S is an oligonucleotide of sufficient length to independently interact stably with a target sequence (the target could be an RNA, DNA or mixed RNA / DNA polymers); and represents a phosphodiester bond.

FORMULA V in which, each P, N, F, V, Z and M independently represent a nucleotide that could be the same or different; • indicates the formation of hydrogen bonds between two adjacent nucleotides; N 'is a complementary nucleotide for N; F 'is a complementary nucleotide for F; or is an integer greater than or equal to 3, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12 or 15; k is zero or is an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20; t is zero or is an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20; w is an integer greater than or equal to 4 and preferably less than about 100, more specifically 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; d is an integer greater than or equal to 3 and preferably less than about 20, more specifically 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, or 18; h is an integer greater than or equal to 2 and preferably less than about 10, more specifically 2, 3, 4, 5, 6, 7, 8, or 9; c is an integer greater than or equal to 0 and preferably less than about 20, more specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, or 18; (P) t, (P) k, (P) t ?, (P) ??, (V), and (Z) c, are oligonucleotides, which preferably include at least one position other than deoxynucleotide (by nucleotide example containing 2'-H); each of (P) t. (P) k. (P) t? > (P) M, (V), and (Z) c, could include linkers of the phosphodiester, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate and the like type or a combination thereof; k and t could have the same length (k = t) or they could have different lengths (k? t); (M) w is a sequence of oligonucleotides whose inter-nucleotide linkers could include linkers of the phosphodiester, phosphorothioate, 5'-thiophosphate, methylphosphonate or phosphorodithioate type, or a combination thereof; t, k and w could have the same length (k = t = w), or they could have different lengths (k? t? w) or (k = t? w) or (k? t = w) or (k = w? t); at least one or more of each said (P) ty (P) k, and (M) w is an oligonucleotide of sufficient length to independently interact stably with a target sequence (the target could be an RNA, a DNA or mixed RNA / DNA polymers); each B and B 'independently represent a blocking structure, which independently could be present or absent; and represents a chemical bond (for example a phosphate ester bond, an amide bond, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate or others known in the art). In a preferred embodiment N and / or N 'in (N * N') 0, F and / or F 'in (F »F') h and / or (Z) c, could optionally be able to interact independently with a target sequence.

FORMULA VI in which, each P, N, F, Z and M independently represent a nucleotide which may be the same or different; • indicates the formation of hydrogen bonds between two adjacent nucleotides, N 'is a nucleotide complementary to N; F 'is a nucleotide complementary to F; or is an integer greater than or equal to 3, more specifically 4, 5, 6, 7, 9, 10, 11, 12, or 15; k is zero or an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20; k1 is zero or an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 15 or 20; t is zero or an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, , 11, 12, 15 or 20; t1 is zero or an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 15 or 20; w is an integer greater than or equal to 4 or preferably less than 100, more specifically 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20; h is an integer greater than or equal to 2 and preferably less than about 10, more specifically 2, 3, 4, 5, 6, 7, 8 or 9; c is an integer greater than or equal to 0 and preferably less than about 20, more specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16 or 18; (P) t, (P) k, and (Z) c is an oligonucleotide that preferably includes at least one position that is not a deoxynucleotide (e.g., nucleotide containing 2'H); each (P) t, (P) k, and (Z) c could include linkers such as phosphodiester, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate and the like or a combination thereof; k and t could be of the same length (k = t) or of different lengths (k? t); k1 and t1 could have the same length (k1 = t1) or different lengths (k1? t1); (M) w is a sequence of oligonucleotides whose nucleotide linkers could include linkers of the phosphodiester, phosphorothioate, 5'-thiophosphate, methylphosphonate or phosphorodithioate type or a combination thereof; t, k, and w, could have the same length (k = t = w) or have different lengths (k? t? w) or (k = t? w) or (k? t = w) or (k = w ? t); t1, k1, and w could have the same length (k1 = t1 = w) or have different lengths (k1? t1? w) or (k1 = t1? w) or (k1? t1 = w) or (k1 = w? t1); at least one or more of each of said (P) p, (P) k, (P) t ?, (P)? i. and (M) w is an oligonucleotide of sufficient length to interact independently in stable form with an objective sequence (the target could be a polymer of RNA, DNA or mixed RNA / DNA polymers); each B and B 'independently represent an end blocking structure that could independently be present or absent; and represents a chemical bond (for example, a phosphate ester bond, an amide bond, a phosphorothioate bond, a phosphorodithioate bond, or others known in the art). In a preferred embodiment N and / or N 'in (N »N') 0, F and / or F 'in (F» F') and / or (Z) c, it may optionally be able to interact independently with an objective sequence.

FORMULA Vil wherein, each P, N, F, V, Z, and M independently represents a nucleotide which may be the same or different; • indicates the formation of hydrogen bonds between two adjacent nucleotides, N 'is a nucleotide complementary to N; F 'is a nucleotide complementary to F; or is an integer greater than or equal to 3, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, or fifteen; k is zero or an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 15 or 20; t is zero or an integer greater than or equal to 3 and preferably less than about 100, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20; w is an integer greater than or equal to 4 and preferably less than 100, more specifically 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20; d is an integer greater than or equal to 3 and preferably less than about 20, more specifically 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, or 18; h is an integer greater than or equal to 2 and preferably less than about 10, more specifically 2, 3, 4, 5, 6, 7, 8, or 9; c is an integer greater than or equal to 0 and preferably less than about 20, more specifically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, or 18; (P) t, (P) k, (V) d and (Z) c are oligonucleotides that preferably include at least one non-deoxynucleotide position (eg nucleotide containing 2'-H); each (P) t, (P) k, (V) d and (Z) c could include linkers of the phosphodiester, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate and the like type or a combination thereof; k and t could have the same length (k = t) or different lengths (k? t); (M) w is an oligonucleotide sequence whose intemucleotide linkers could include linkers of phosphodiester phosphorothioate, 5'-thiophosphate, methylphosphonate or phosphorodithioate type or a combination thereof; t, k, and w could have the same length (k = t = w) or have different lengths (k? t? w) or (k = t? w) or (k? t = w) or (k = w? t); at least one or more of each of said (P) t, (P) k, and (M) w is a oligonucleotide of sufficient length to stably interact independently with a target sequence (the target could be RNA, DNA or mixed RNA / DNA polymers); each B and B 'independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond (for example a phosphate ester bond, an amide bond, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate or others known in the art). In a preferred embodiment N and / or N 'in (N * N') 0, F and / or F 'in (F * F') and / or (Z) c, they could optionally be able to interact independently with an objective sequence.

FORMULA VIII B I B "I F | D I - 0 I I K - - r \ / w wherein, each F, D, O, K, W, and T independently represents an oligonucleotide whose nucleotide sequence could be the same or different; they could be of the same length or of different length and could include linkers of the phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, methylphosphonate and the like type or a combination thereof; F and D independently or in combination form an RNAse activation domain H, wherein F and D are greater than or equal to 4 nucleotides in length if they form independent RNAse H domains and are greater than or equal to 2 nucleotides in length if they form RNAse H domains in combination; • indicates the formation of hydrogen bonds between two adjacent nucleotides within an oligonucleotide; D comprises the nucleotide sequence that is complementary to the nucleotide sequence of O; K comprises the nucleotide sequence that is complementary to the nucleotide sequence of T; D and O form base pairs greater than or equal to two with each other which are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 Base pairs; K and T form base pairs greater than or equal to two with each other which are contiguous or non-contiguous, more specifically K and T form 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 Base pairs; the regions of paired bases K »T and O« D could be contiguous or non-contiguous with each other; K, T, O and W are of length greater than or equal to 3 nucleotides and preferably less than about 100 nucleotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20; F, D, K, T, W and O together are of sufficient length to interact stably with a target nucleic acid sequence (the target may be RNA, DNA or mixed RNA / DNA polymers); each B and B 'independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond (for example a phosphate ester bond, an amide bond, phosphorothioate, 5'-thiophosphate, phosphorodithioate, methylphosphonate or others known in the art).

FORMULA IX wherein, each F, D, O, K, W, and T independently represents an oligonucleotide whose nucleotide sequence could be the same or different; they could be of the same length or of different length and could include linkers of the phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, methylphosphonate and the like type or a combination thereof; F and D independently or in combination form an RNAse H activation domain, wherein F and D are of greater than or equal to 4 nucleotides; • indicates the formation of hydrogen bonds between two adjacent nucleotides within an oligonucleotide; D comprises the nucleotide sequence that is complementary to the nucleotide sequence of O; K comprises the nucleotide sequence that is complementary to the nucleotide sequence of T; D and O form larger or equal pairs of bases with two that are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 base pairs; K and T form base pairs greater than or equal to two with each other which are contiguous or non-contiguous, more specifically K and T form 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 Base pairs; the regions of paired bases K »T and O» D could be contiguous or non-contiguous one with another; K, T, O and W are of length greater than or equal to 3 nucleotides and preferably less than about 100 nucleotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20; F, D, K, T, W and O together are of sufficient length to interact stably with a target nucleic acid sequence (the target may be RNA, DNA or mixed RNA / DNA polymers); each B and B 'independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond (for example a phosphate ester bond, an amide bond, 5'-thiophosphate, phosphorothioate, phosphorodithioate, methylphosphonate or others known in the art).

FORMULA X B B "I F I O | - D I I I K - T \ / w in which, each F, D, O, K, W, and T independently represents an oligonucleotide whose nucleotide sequence could be the same or different; they could be of the same length or of different length and could include linkers of the phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, methylphosphonate and the like type or a combination thereof; F and D so independently form an RNAse H activation domain, where F and D are of greater than or equal to 4 nucleotides; • indicates the formation of hydrogen bonds between two adjacent nucleotides within an oligonucleotide; D comprises the nucleotide sequence that is complementary to the nucleotide sequence of O; K comprises the nucleotide sequence that is complementary to the nucleotide sequence of T; D and O form base pairs greater than or equal to two with each other which are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 Base pairs; K and T form base pairs greater than or equal to two with each other which are contiguous or non-contiguous, more specifically K and T form 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 Base pairs; the paired base regions K »T and 0 * D could be contiguous or non-contiguous with one another; K, T, O and W are of length greater than or equal to 3 nucleotides and preferably less than about 100 nucleotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20; F, D, K, T, W and O together are of sufficient length to interact stably with a target nucleic acid sequence (the target may be RNA, DNA or mixed RNA / DNA polymers); each B and B 'independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond (for example a phosphate ester bond, an amide bond, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate or others known in the art).

FORMULA XI wherein, each F, D, O, K, W, and T independently represents an oligonucleotide whose nucleotide sequence could be identical or different; they could be of the same length or of different length and could include linkers of the phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, methylphosphonate and the like type or a combination thereof; F and D independently or in combination form an activation domain of RNase H, where F and D are greater than or equal to 4 nucleotides in length if they form independent RNase H domains and are greater than or equal to 2 nucleotides in length if form RNAse H domains in combination; • indicates the formation of hydrogen bonds between two adjacent nucleotides within an oligonucleotide; D comprises the nucleotide sequence that is complementary to the nucleotide sequence of O; K comprises the nucleotide sequence that is complementary to the nucleotide sequence of T; D and O form larger or equal pairs of bases with two that are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 base pairs; K and T form base pairs greater than or equal to two with each other that are contiguous or non-contiguous, in a more specific K and T form 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 base pairs; the paired base regions K »T and O * D could be contiguous or non-contiguous with each other; K, T, O and W are of length greater than or equal to 3 nucleotides and preferably less than about 100 nucleotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20; F, D, K, T, W and O together are of sufficient length to interact stably with a target nucleic acid sequence (the target may be RNA, DNA or mixed RNA / DNA polymers); each B and B 'independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond (for example a phosphate ester bond, an amide bond, phosphorothioate, 5'-thiophosphate, phosphorodithioate, methylphosphonate or others known in the art).

FORMULA XII B B 'D - O W wherein, each D, O, and W independently represents an oligonucleotide whose nucleotide sequence could be the same or different; they could be of the same length or of different length and could include linkers of the phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate and similar or a combination thereof; D independently forms an RNAse H activation domain of greater than or equal to 4 nucleotides; • indicates the formation of hydrogen bonds between two adjacent nucleotides within an oligonucleotide; D comprises the nucleotide sequence that is complementary to the nucleotide sequence of O; D and O form base pairs greater than or equal to two with each other which are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 Base pairs; O and W are of greater length than or equal to 3 nucleotides and preferably less than about 100 nucleotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or twenty; D, W and O together are of sufficient length to interact stably with a target nucleic acid sequence (the target may be RNA, DNA or mixed RNA / DNA polymers); each B and B 'independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond (for example a phosphate ester bond, an amide bond, phosphorothioate, phosphorodithioate, methylphosphonate or others known in the art).

FORMULA XIII B B '1 I O- D w in which, each D, O, and W independently represents an oligonucleotide whose nucleotide sequence could be the same or different; they could be of the same length or of different length and could include linkers of the phosphodiester, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate and the like type or a combination thereof; D independently forms an RNAse H activation domain of greater than or equal to 4 nucleotides; • indicates the formation of hydrogen bonds between two adjacent nucleotides within an oligonucleotide; D comprises the nucleotide sequence that is complementary to the nucleotide sequence of O; D and O form base pairs greater than or equal to two with each other which are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 Base pairs; O and W are of greater length than or equal to 3 nucleotides and preferably less than about 100 nucleotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or twenty; D, W and O together are of sufficient length to interact stably with a target nucleic acid sequence (the target may be RNA, DNA or mixed RNA / DNA polymers); each B and B 'independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond (for example a phosphate ester bond, an amide bond, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate or others known in the art).

FORMULA XIV B B 'I A I O - D I I K - T \ / w wherein, each A, D, O, K, W, and T independently represents an oligonucleotide whose nucleotide sequence could be the same or different; it could be of the same length or of different length and could include linkers of the phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, methylphosphonate and the like type or a combination thereof; D independently forms an RNAse H activation domain in length greater than or equal to 4 nucleotides; • indicates the formation of hydrogen bonds between two adjacent nucleotides within an oligonucleotide; D comprises the nucleotide sequence that is complementary to the nucleotide sequence of O; K comprises the nucleotide sequence that is complementary to the nucleotide sequence of T; D and O form pairs of bases greater than or equal to two with one another which are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 base pairs; K and T form base pairs greater than or equal to two with each other which are contiguous or non-contiguous, more specifically K and T form 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 Base pairs; the base regions Paired KVT and 0 * D could be contiguous or not contiguous with each other; A, K, T, O and W are of length greater than or equal to 3 nucleotides and preferably less than about 100 nucleotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20; A, D, K, T, W and O together are of sufficient length to interact stably with a target nucleic acid sequence (the target may be RNA, DNA or mixed RNA / DNA polymers); each B and B 'independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond (for example a phosphate ester bond, an amide bond, phosphorothioate, 5'-thiophosphate, phosphorodithioate, methylphosphonate or others known in the art).

FORMULA XV B B 'I A I O • D I I K - T V wherein, each A, D, O, K, W, and T independently represents an oligonucleotide whose nucleotide sequence could be the same or different; it could be of the same length or of different length and could include linkers of the phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, methylphosphonate and the like type or a combination thereof; D independently forms an RNAse H activation domain of greater than or equal to 4 nucleotides; • indicates the formation of hydrogen bonds between two adjacent nucleotides within an oligonucleotide; D comprises the nucleotide sequence that is complementary to the nucleotide sequence of O; K comprises the nucleotide sequence that is complementary to the nucleotide sequence of T; D and O form base pairs greater than or equal to two with each other which are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 Base pairs; K and T form base pairs greater than or equal to two with each other which are contiguous or non-contiguous, more specifically K and T form 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 Base pairs; the paired base regions K T and OD could be contiguous or non-contiguous with one another; A, K, T, O and W are of length greater than or equal to 3 nucleotides and preferably less than about 100 nucleotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20; A, D, K, T, W and O together are of sufficient length to interact stably with a target nucleic acid sequence (the target may be RNA, DNA or mixed RNA / DNA polymers); each B and B 'independently represent a blocking structure of end which independently could be present or absent; and represents a chemical bond (for example a phosphate ester bond, an amide bond, phosphorothioate, 5'-thiophosphate, phosphorodithioate, methylphosphonate or others known in the art).

FORMULA XVI wherein, each A, D, O, K, W, and T independently represents an oligonucleotide whose nucleotide sequence could be the same or different; it could be of the same length or of different length and could include linkers of the phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, methylphosphonate and the like type or a combination thereof; D independently forms an RNAse H activation domain of greater than or equal to 4 nucleotides; • indicates the formation of hydrogen bonds between two adjacent nucleotides within an oligonucleotide; D comprises the nucleotide sequence that is complementary to the nucleotide sequence of O; K comprises the nucleotide sequence that is complementary to the nucleotide sequence of T; D and O form greater or equal pairs of bases to two with each other that are contiguous or not contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 base pairs; K and T form larger or equal pairs of bases to two with each other that are contiguous or non-contiguous, more specifically K and T form 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 base pairs; the regions of paired bases K «T and O« D could be contiguous or not contiguous with each other; A, K, T, O and W are of length greater than or equal to 3 nucleotides and preferably less than about 100 nucleotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20; A, D, K, T, W and O together are of sufficient length to interact stably with a target nucleic acid sequence (the target may be RNA, DNA or mixed RNA / DNA polymers); each B and B 'independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond (for example, a phosphate ester bond, an amide bond, phosphorothioate, phosphorodithioate, methylphosphonate, 5'-thiophosphate, or others known in the art).

FORMULA XVII in which, each A, D, O, K, W, and T independently represents a oligonucleotide whose nucleotide sequence could be the same or different; it could be of the same length or of different length and could include linkers of the phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, methylphosphonate and the like type or a combination thereof; D independently forms an RNAse H activation domain of greater than or equal to 4 nucleotides; • indicates the formation of hydrogen bonds between two adjacent nucleotides within an oligonucleotide; D comprises the nucleotide sequence that is complementary to the nucleotide sequence of O; K comprises the nucleotide sequence that is complementary to the nucleotide sequence of T; D and O form larger or equal pairs of bases with two that are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 base pairs; K and T form larger or equal pairs of bases to two with each other that are contiguous or non-contiguous, more specifically K and T form 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 base pairs; the paired base regions K »T and 0» D could be contiguous or non-contiguous with one another; A, K, T, O and W are of length greater than or equal to 3 nucleotides and preferably less than about 100 nucleotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20; A, D, K, T, W and O together are of sufficient length to interact stably with a target nucleic acid sequence (the target may be RNA, DNA or mixed RNA / DNA polymers); each B and B 'independently represent an end blocking structure which independently could be present or absent; Y represents a chemical bond (for example, a phosphate ester bond, an amide bond, phosphorothioate, 5'-thiophosphate, phosphorodithioate, methylphosphonate, or others known in the art).

FORMULA XVIII B B 'I A O- D W wherein, each A, D, O, and W independently represents an oligonucleotide whose nucleotide sequence could be the same or different; it could be of the same length or different length and could include linkers of the phosphodiester, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methyphosphonate and the like type or a combination thereof; D independently forms an RNAse H activation domain of greater than or equal to 4 nucleotides; • indicates the formation of hydrogen bridges between two adjacent nucleotides within an oligonucleotide; D comprises the nucleotide sequence that is complementary to the nucleotide sequence of O; D and O form base pairs greater than or equal to two with each other which are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 Base pairs; A, O and W are of length greater than or equal to 3 nucleotides and preferably less than about 100 nucleotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20; A, D, W and O together are of sufficient length to interact stably with a target nucleic acid sequence (the target may be RNA, DNA or mixed RNA / DNA polymers); each B and B 'independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond (for example, a phosphate ester bond, an amide bond, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate, or others known in the art).

FORMULA XIX wherein, each A, D, O, and W independently represents an oligonucleotide whose nucleotide sequence could be the same or different; it could be of the same length or of different length and could include linkers of the phosphodiester, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate and the like type or a combination thereof; D independently forms an RNAse H activation domain in length greater than or equal to 4 nucleotides; • indicates the formation of hydrogen bridges between two adjacent nucleotides within an oligonucleotide; D comprises the nucleotide sequ that is complementary to the nucleotide sequ of O; D and O form base pairs greater than or equal to two with each other which are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 Base pairs; A, O and W are of length greater than or equal to 3 nucleotides and preferably less than about 100 nucleotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20; A, D, W and O together are of sufficient length to interact stably with a target nucleic acid sequ (the target may be RNA, DNA or mixed RNA / DNA polymers); each B and B 'independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond (for example, a phosphate ester bond, an amide bond, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate, or others known in the art).

FORMULA XX B B 'I F O D W wherein, each A, D, O, and W independently represents an oligonucleotide whose nucleotide sequ could be the same or different; it could be of the same length or of different length and could include linkers of the phosphodiester, phosphorothioate, phosphorodithioate, 5'-thiophosphate, and / or methylphosphonate type and the like or a combination thereof; F and D independently form RNAse H activation domains greater than or equal to 4 nucleotides; • indicates the formation of hydrogen bonds between two adjacent nucleotides within an oligonucleotide; D comprises the nucleotide sequ that is complementary to the nucleotide sequ of O; D and O form base pairs greater than or equal to two with each other which are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 Base pairs; O and W are of greater length than or equal to 3 nucleotides and preferably less than about 100 nucleotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or twenty; F, D, W and O together are of sufficient length to interact stably with a target nucleic acid sequ (the target may be RNA, DNA or mixed RNA / DNA polymers); each B and B 'independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond (for example a phosphate ester bond, an amide bond, phosphorothioate, 5'-thiophosphate, phosphorodithioate, methylphosphonate, or others known in the art).

FORMULA XXI B B 'F O D W wherein, each F, D, O, and W independently represents an oligonucleotide whose nucleotide sequ could be the same or different; it could be of the same length or of different length and could include linkers of the phosphodiester, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate and the like type or a combination thereof; F and D independently or in combination form RNAse H activation domains, where F and D are greater than or equal to 4 nucleotides if they form RNAse H activation domains and are greater than or equal to 2 nucleotides if they form RNAse H activation domains in combination; • indicates the formation of hydrogen bonds between two adjacent nucleotides within an oligonucleotide; D comprises the nucleotide sequ that is complementary to the nucleotide sequ of O; K comprises the nucleotide sequ that is complementary to the nucleotide sequ of T; D and O form base pairs greater than or equal to two with each other which are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 Base pairs; K and T form base pairs greater than or equal to two with each other that are contiguous or non-contiguous, more specifically K and T form 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 base pairs; the base-paired regions K * T and O * D they could be contiguous or not contiguous with each other; O and W are of greater length than or equal to 3 nucleotides and preferably less than about 100 nucleotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or twenty; F, D, W and O together are of sufficient length to interact stably with a target nucleic acid sequence (the target may be RNA, DNA or mixed RNA / DNA polymers); each B and B 'independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond (for example a phosphate ester bond, an amide bond, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate or others known in the art).

FORMULA XXII B B 'I A D - O W wherein, each A, D, O, and W independently represents an oligonucleotide whose nucleotide sequence could be the same or different; could be of the same length or different length and could include linkers of the phosphodiester, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate and the like type or a combination thereof; D independently forms an RNAse H activation domain of greater than or equal to 4 nucleotides; • indicates the formation of hydrogen bonds between two adjacent nucleotides within an oligonucleotide; D comprises the nucleotide sequence that is complementary to the nucleotide sequence of O; D and O form base pairs greater than or equal to two with each other which are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 Base pairs; A, O and W are of length greater than or equal to 3 nucleotides and preferably less than about 100 nucleotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20; A, D, W and O together are of sufficient length to interact stably with a target nucleic acid sequence (the target may be RNA, DNA or mixed RNA / DNA polymers); each B and B 'independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond (for example, a phosphate ester bond, an amide bond, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate, or others known in the art).

FORMULA XXIII wherein, each A, D, O, and W independently represents an oligonucleotide whose nucleotide sequence could be the same or different; it could be of the same length or of different length and could include linkers of the phosphodiester, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate and the like type or a combination thereof; D independently forms an RNAse H activation domain of greater than or equal to 4 nucleotides; • indicates the formation of hydrogen bonds between two adjacent nucleotides within an oligonucleotide; D comprises the nucleotide sequence that is complementary to the nucleotide sequence of O; D and O form larger or equal pairs of bases with two that are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 base pairs; A, O and W are of length greater than or equal to 3 nucleotides and preferably less than about 100 nucleotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 20; A, D, W and O together are of sufficient length to interact stably with a target nucleic acid sequence (the target may be RNA, DNA or mixed RNA / DNA polymers); every B and B ' independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond (for example, a phosphate ester, 5'-thiophosphate bond, an amide bond, phosphorothioate, phosphorodithioate, methylphosphonate, or others known in the art).

FORMULA XXIV B B I F D -O W wherein, each F, D, O, and W independently represents an oligonucleotide whose nucleotide sequence could be the same or different; it could be of the same length or of different length and could include linkers of the phosphodiester, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate and the like type or a combination thereof; F and D independently or in combination form RNAse H activation domains, where F and D are greater than or equal to 4 nucleotides if they form RNAse H activation domains and are greater than or equal to 2 nucleotides if they form RNAse H activation domains in combination; • indicates the formation of hydrogen bonds between two adjacent nucleotides within an oligonucleotide; D understands the nucleotide sequence that is complementary to the nucleotide sequence of O; K comprises the nucleotide sequence that is complementary to the nucleotide sequence of T; D and O form base pairs greater than or equal to two with each other which are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 Base pairs; K and T form base pairs greater than or equal to two with each other which are contiguous or non-contiguous, more specifically K and T form 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 Base pairs; the regions of paired bases K «T and O * D could be contiguous or not contiguous with each other; O and W are of greater length than or equal to 3 nucleotides and preferably less than about 100 nucleotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or twenty; F, D, W and O together are of sufficient length to interact stably with a target nucleic acid sequence (the target may be RNA, DNA or mixed RNA / DNA polymers); each B and B 'independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond (for example a phosphate ester, 5'-thiophosphate bond, an amide bond, phosphorothioate, phosphorodithioate, methylphosphonate or others known in the art).

FORMULA XXV B B I I F D -0 W wherein, each A, D, O, and W independently represents an oligonucleotide whose nucleotide sequence could be the same or different; it could be of the same length or of different length and could include linkers of the phosphodiester, phosphorothioate, phosphorodithioate, 5'-thiophosphate, methylphosphonate and the like type or a combination thereof; F and D independently form RNAse H activation domains greater than or equal to 4 nucleotides; • indicates the formation of hydrogen bonds between two adjacent nucleotides within an oligonucleotide; D comprises the nucleotide sequence that is complementary to the nucleotide sequence of O; D and O form base pairs greater than or equal to two with each other which are contiguous or non-contiguous, more specifically D and O form 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 Base pairs; O and W are of greater length than or equal to 3 nucleotides and preferably less than about 100 nucleotides, more specifically 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or twenty; F, D, W and O together are of sufficient length to interact stably with a target nucleic acid sequence (the target may be RNA, DNA or mixed RNA / DNA polymers); each B and B 'represent in a way independent an end blocking structure which could independently be present or absent; and represents a chemical bond (e.g., a phosphate ester bond, an amide bond, phosphorothioate, phosphorodithioate, d-thiophosphate, methylphosphonate, or others known in the art). In a preferred embodiment, the invention features an HPH molecule of any of formulas l-III, and V-VII, wherein (M) w optionally includes a RNase-H activation region. With "activation region" of RNase H "or" RNAse H Activation Region ", is meant, a region (generally of more than or equal to 4 nucleotides long, preferably 5, 6, 7, 8, 9, 10, or 11 nucleotides) of a nucleic acid molecule that can bind to an RNA objective to form, for example, a complex (M) W "target RNA that is recognized by the cellular RNase H enzyme, where the RNAse H enzyme will then bind to the (M) W" target RNA complex and cut the target sequence. The RNase H activation region comprises phosphodiester base structure chemistry, phosphorothioate (preferably four of the nucleotides are phosphorothioate substitutions); more specifically, either 4, 5, 6, 7, 8, 9, 10, or 11 of the nucleotides are phosphorothioate substitutions), phosphorodithioate, 5'-thiophosphate, or methylphosphonate or a combination thereof. In addition to one or more of the above-described base structure chemistries, the RNAse H activation region comprises deoxyribose, arabino, fluoroarabino or a combination thereof, and nucleotide sugar chemistry.

Those skilled in the art will recognize that the foregoing are non-limiting examples and that any combination of phosphate, sugar and base chemistry of a nucleic acid that supports the activity of the RNase H enzyme is within the scope of the definition of the region of RNAse H activation and the scope of the present invention. "Nucletide" as used in the present invention, as recognized in the art, includes natural (standard) bases, and modified bases that are well known in the art. Such bases are usually located at the 1 'position of a portion of nucleotide sugar. The nucleotides usually comprise a base, a sugar and a phosphate group. The nucleotides may be unmodified or may be modified in the sugar, phosphate and / or base moiety, (also referred interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and others; Example Usman and McSwiggen, supra; Eckstein et al., PCT International Publication No. WO 92/07065; Usman et al., PCT International Publication No. WO 93/15187; Uhlman and Peyman, supra) of which all are incorporated in the present invention for reference. Examples of modified nucleic acid bases are known in the art and have been recently summarized by Limbach et al., 1994, Nucleic Acid Res. 22,2183. Some of the non-limiting examples of base modifications that can be introduced into the nucleic acid molecules include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6- trimethoxybenzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (for example, 5-methylcytidine), 5-alkyluridines (for example, ribotimidine), 5-halogenouridine (for example, 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (for example, 6-methyluridine), propyne and others (Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman and Peyman, supra). With "modified bases" in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil in the V-position or their equivalents; such bases could be used in any position, for example, within the catalytic center of an enzymatic nucleic acid molecule and / or in the substrate binding regions of the nucleic acid molecule. By "ribonucleotide" is meant a nucleotide with one of the bases adenine, cytosine, guanine or uracil linked to the 1 'carbon of the β-D-ribo-furanose. By "unmodified nucleotide" is meant a nucleotide with one of the bases adenine, cytosine, guanine or uracil linked to the 1 'carbon of the β-D-ribofuranose. By "modified nucleotide" is meant a nucleotide containing a modification in the chemical structure of a base, sugar and / or phosphate of an unmodified nucleotide. By "abasic" is meant portions of nucleic acid sugar that lack a base or that have other chemical groups instead of a base in the V position.

By "sufficient length" is meant generally an oligonucleotide larger than or equal to 4 nucleotides, or an equivalent chemical moiety that can be linked to interact with a target nucleic acid molecule in solution and / or in a cell under physiological conditions. By "complementarity" is meant that a nucleic acid can form a bridge or hydrogen bonds with another RNA sequence by the traditional type of Watson-Crick or other non-traditional types. With reference to the nucleic acid molecules of the present invention, the binding free energy for a nucleic acid molecule with its target or complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, for example, ribozyme cleavage, inhibition of antisense or triple helix. The determination of binding free energies for nucleic acid molecules is well known in the art (see, for example, Turner et al., 1987, CSH Symp. Quant. Biol. Lll pp 123-133; Frier et al. , 1986, Proc. Nat. Acad. Sci. USA 83: 9373-9377; Turner et al., 1987, J. Am. Chem. Soc. 109: 3783-3785. A percentage of complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (eg, base pairing of Watson-Crick type) with a second nucleic acid sequence (eg, 5, 6, 7). , 8, 9, 10 of 10 being 50%, 60%, 70%, 80%, 90% and 100% complementary). "Perfectly complementary" means that all contiguous residues of a nucleic acid sequence will be bound by hydrogen bonds with the same number of contiguous residues in a second nucleic acid sequence. By "stably interacting" is meant an interaction of the oligonucleotides with the target nucleic acid (e.g., forming hydrogen bonds with the complementary nucleotides in the target under physiological conditions). The term should also mean the interaction of HPH molecules with the target molecule for a sufficient time, under physiological conditions, in solution or in a cell, so that the HPH molecule interferes with the function of the target nucleic acid molecule. Typically, the antisense molecules will be complementary to an objective sequence along a single contiguous sequence of the antisense molecule. However, in some embodiments, an antisense molecule could be attached to the substrate in such a way that the substrate molecule forms a loop, and / or the antisense molecule could be joined in such a way that the antisense molecule forms a loop. Therefore, the antisense molecule could be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule could be complementary to an objective sequence or both. With "nucleic acid molecule" as used in the present invention means a molecule constituted by nucleotides. The nucleic acid may be constituted by modified or unmodified nucleotides or by non-nucleotides or by various mixtures and combinations thereof.

By "inhibit" is meant that the activity of the target genes or the level of mRNA molecules or equivalent mRNA molecules coding for the target genes is reduced below the level observed in the absence of the nucleic acid molecule of the target genes. the invention. In one embodiment, the inhibition with HPH molecules is preferably below the level observed in the presence of an unpaired nucleic acid molecule that can not stably bind to the same site on the mRNA. In another embodiment, the inhibition with HPH molecules is preferably greater than that observed in the presence of, for example, an oligonucleotide with a mixed sequence or non-matings. In another embodiment, the inhibition of the target genes with the nucleic acid molecule of the present invention is greater in the presence of the nucleic acid molecule than in its absence. By "inhibiting" is also meant an impediment to the normal function of a macromolecule caused by the introduction of a foreign substance, such as the HPH molecule. By "target sequence" or "target nucleic acid molecule" is meant a gene or a partial sequence thereof, and those elements necessary for its expression, regulation or its transcription or its product or replication intermediates or portions thereof, including DNA, RNA or protein. Non-limiting examples of the targeted mRNA sequence of c-raf, hepatitis C RNA, endothelial growth factor receptor (e.g., flt- and KDR), ras RNA and the like. With "gene" is meant a nucleic acid that codes for a RNA By "antisense" is meant a nucleic acid molecule that binds to the target RNA by means of RNA-RNA or RNA-DNA or RNA-ANP interactions (Nucleic Acid of Protein; Egholm et al., 1993, Nature 365, 566 ) and alters the activity of the target RNA (for a review see Stein and Cheng, 1993, Science 261, 1004). With RNA "equivalent" to the target genes it is meant that those RNA molecules present in nature that have homology (partial or complete) to the genes or that encode proteins with function similar to that of the genes in various animals are included. , including humans, rodents, primates, rabbits and pigs. The equivalent RNA sequence also includes, in addition to the coding region, regions such as the 5 'untranslated region, the 3' untranslated region, introns, intron-exon junctions and the like. By "related (a)" it is meant that the inhibition of the RNA molecules for the target gene and therefore the reduction in the respective levels of protein activity will alleviate to some degree the symptoms of the disease or condition. By "blocking structures" it is meant chemical modifications that have been incorporated in the terminal end of the oligonucleotide (for example B and B 'in the above formulas). These modifications at the terminal ends protect the nucleic acid molecule from degradation with exonuclease, and could help supply and / or location inside a cell. In another embodiment, (P) k, (P) t, (N.N ') or, (F.F') h, (V) d, (Z) c, (P) k1, (P) t1, (M) w, (Y) r, (Y) f, D, K, T, W and / or E, independently include modifications that are selected from a group comprising 2'-O-alkyl (e.g., 2'-0-allyl, Sproat et al., Supra) sometimes referred to as RNA modifications; 2'-0-alkylthioalkyl (for example 2'-0-methylthiomethyl; Karpeisky et al., 1998, Nucleosides &Nucleotides 16, 955-958); L-nucleotides (Tazawa et al., 1970, Biochemistry 3499, Ashley, 1992, J. Am. Chem. Soc. 114, 9731; Klubman et al., 1996, Nature Biotech 14, 1112); 2'-C-alkyl (Beigelman et al., 1995, J. Biol. Chem. 270, 25702); 1-5-anhydrohexitol; 2,6-diaminopurine (Strobel et al., 1994, Biochem.33, 13824-13835); 2 '- (N-alanyl) amino-2'-deoxynucleotide; 2 '- (/ V-beta-alanyl) amino-2'-deoxy-2'- (lysyl) amino; 2'-0-amino (Karpeisky et al., 1995, Tetrahedron Lett 39, 1131); 2'-deoxy-2 '- (A / -histidyl) amino; 5-methyl (Strobel, supra); 2 '- (? / - b-carboxamidin-beta-alanyl) amino; 2'-deoxy-2 '- (N-beta-alanyl) (Matulic-Adamic et al., 1995, Bioorg. & Med. Chem. Lett., 5, 2721-2724); xylofuranosyl (Rosemeyer et al., 1991, Helvética Chem. Acta, 74, 748; Seela et al., 1994, Helvética Chem. Acta, 77, 883; Seela et al., 1996, Helvética Chem. Acta, 79, 1451) . Even in another embodiment, B 'is selected from a group comprising inverted abbasic residue, 4' nucleotide, 5'-methylene, nucleotide 1- (beta-D-eritrofuranosyl), nucleotide 4'-thio, carbocyclic nucleotide; nucleotide 1,5-anhydrohexitol; L-nucleotides; alpha-nucleotides; nucleotide of modified base; phosphorodithioate linkage; Ireo-pentofuranosyl nucleotide; 3 ', 4'-non-cyclic dry nucleotide; non-cyclic 3,4-dihydroxybutyl nucleotide; 3,4-dihydroxypentyl nucleotide non-cyclic; 3'-3'-inverted nucleotide portion; 3'-3'-inverted abasic portion; 3'-2'-inverted nucleotide portion; abasic portion 3'-2'-n poured; 1,4-butanediol phosphate; 3'-phosphoramidate; hexyl phosphate; aminohexylphosphate; 3'-phosphate; phosphorothioate (preferably three of the terminal nucleotides are phosphorothioate substitutions, more specifically, any of 1, 2, 3, 4, or 5 of the terminal nucleotides are phosphorothioate substitutions); phosphorodithioate; or a portion of bridge-forming or non-bridge-forming methylphosphonate (for more details see Beigelman et al., PCT International Publication No. WO 97/26270, incorporated herein by reference). Even in another embodiment, B is selected from a group comprising nucleotide 4 ', 5'-methylene, nucleotide I- (beta-D-eritrofuranosyl), nucleotide 4'-thio, carbocyclic nucleotide; 5'-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1, 2-aminododecyl phosphate; hydroxypropyl phosphate; 1, 5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; nucleotide of modified base; phosphorodithioate; Ireo-pentofuranosyl nucleotide; 3 ', 4'-non-cyclic dry nucleotide; 3,4-dlhydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide; 5'-5'-inverted nucleotide portion; 5'-5'-inverted abasic portion; 5'-phosphoramidate; 5'-phosphorothioate; 1,4-butanediol phosphate; 5'-amino; 5'-phosphoramidate bridging or non-bridging; phosphorothioate (preferably three of the terminal nucleotides are phosphorothioate substitutions; specific, any of 1, 2, 3, 4, or 5 of the terminal nucleotides are phosphorothioate substitutions) and / or phosphorodithioate, portions of methylphosphonate and d-mercapto bridging or non-bridging (for more details see Beaucage et al. Iyer, 1993, Tetrahedron, 49, 1925; incorporated in the present invention for reference). An "alkyl" group refers to a saturated aliphatic hydrocarbon, including straight chain, branched chain and cyclic alkyl groups. Preferably, the alkyl group has from 1 to 12 carbons. More preferred is a lower alkyl having from 1 to 7 carbons, more preferred still has 1 to 4 carbons. The alkyl group may be substituted or unsubstituted. When substituted, the group or groups preferably substituted are hydroxyl, cyano, akoxy, = 0, = S, NO2, or N (CH3) 2, amino or SH. The term also includes alkenyl groups which are unsaturated hydrocarbon groups containing at least one carbon-carbon double bond, including straight chain, branched chain or cyclic groups. Preferably, the alkenyl group has from 1 to 12 carbons. More preferred is a lower aikenyl having 1 to 7 carbons, more preferred still has 1 to 4 carbons. The alkenyl group may be substituted or unsubstituted. When substituted, the group or groups preferably substituted are hydroxyl, clade, alkoxy, = 0, = S, NO2, halogen, N (CH3) 2, amino or SH. The term "alkyl" also includes alkynyl groups which have an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, including straight chain, branched chain or cyclic groups.

Preferably, the alkynyl group has from 1 to 12 carbons. More preferred is a lower alkynyl having 1 to 7 carbons, more preferred still has 1 to 4 carbons. The alkynyl group may be substituted or unsubstituted. When substituted, the group or groups preferably substituted are hydroxyl, cyano, alkoxy, = 0, = S, NO2, or N (CHs) 2 > amino or SH. Such alkyl groups could also include aryl, alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester groups. An "aryio" group refers to an aromatic group which has at least one ring having a p-conjugated electron system and includes carbocyclic aryl, heterocyclic aryl, and biaryl groups, of which all may be optionally substituted. The preferred substituent or substituents of the aryl groups are halogen, trihalogenomethyl, hydroxyl, SH, OH, cyano, alkoxy, alkyl, alkenyl, alkynyl and amino groups. An "alkylaryl" group refers to an alkyl group (as described above) linked by covalent bond to an aryl group (as described above). The carbocyclic aryl groups are groups in which the ring atoms in the aromatic ring are all carbon atoms. The carbon atoms are optionally substituted. The heterocyclic aryl groups are groups having from 1 to 3 heterogeneous atoms as ring atoms in the aromatic ring and the rest of the ring atoms are carbon atoms. Suitable heterogeneous atoms include oxygen, sulfur, and nitrogen, and include furanyl, thienyl, pyridium, pyrrolyl, N-lower alkylpyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally substituted. An "amide" refers to a -C (O) -NH-R moiety, in which R can be alkyl, aryl, alkylaryl or hydrogen. An "ester" refers to a -C (O) -OR'- moiety, in which R can be alkyl, aryl, alkylaryl or hydrogen. In a preferred embodiment, HPH molecules including the molecules described in the formulas l-XXV can bind to the target nucleic acid molecules in a sequence-specific manner. The stable interaction between the HPH molecule and the target molecules interferes with the normal function of the target molecule. Such an interaction, for example, could cause the inhibition of the function of the target molecule, such as transcription, translation and replication. The HPH molecules of the invention interact with the target molecules and interfere with them in vitro or in vivo in a bacterial cell, in a microbial system, a plant system or a mammalian system to modulate the function of the target molecule in such biological systems. In a preferred embodiment, the HPH molecules of the present invention are used to inhibit the expression of the target gene in a biological system, more specifically in a cell, tissue, organ and animal. In a preferred embodiment, the HPH nucleic acid molecules including the molecules described in the formulas I-III and V-XXV comprise at least one phosphate modification in the base structure, wherein said modification is phosphorothioate base structure chemical (preferably four of the nucleotides have phosphorothioate substitutions, more specifically, any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 17, 19, 21, 23 or 25 of the nucleotides have phosphorothioate substitutions), phosphorodithioate, 5'-thiophosphate or methylphosphonate or a combination thereof. In a preferred embodiment, the HPH nucleic acid molecules including the molecules described in the formulas l-XXV are directly added, or they may be complexed with cationic lipids, packed with liposomes, or otherwise delivered to the target cells. The nucleic acid or nucleic acid complexes can be administered locally to the relevant tissues ex vivo, or in vivo through injection, pump or stent for infusion, with or without its incorporation in biopolymers. In another aspect of the invention, the HPH nucleic acid molecules described in formula IV are expressed from the transcription units inserted into DNA or RNA vectors. Recombinant vectors are preferably DNA plasmids or viral vectors. Viral vectors expressing the HPH molecule could be constructed based on, but not limited to, adeno-associated viruses, retroviruses, adenoviruses, or alphaviruses. Preferably, recombinant vectors that can express the HPH molecules are delivered as described above, and persist in the target cells. Alternatively, viral vectors that ensure transient expression of HPH nucleic acid molecules could be used. Such vectors could be administered repetitively as necessary. Once expressed, the molecules of nucleic acid bind to the target mRNA. The delivery of vectors expressing nucleic acid molecules could be systemic, for example by intravenous or intramuscular administration, by administration to the target cells excised from a patient followed by reintroduction to the patient, or by any other means allowing the introduction into the desired target cell (for a review see Couture and Stinchcomb, 1996, TIG., 12, 510). In another aspect of the invention, nucleic acid molecules that bind target molecules and inhibit cell proliferation are expressed from transcription units inserted into DNA, RNA or viral vectors. Preferably, recombinant vectors that can express HPH molecules are delivered locally as described above, and persistently persist in smooth muscle cells. For this purpose, however, other vectors could be used for mammalian cells that direct RNA expression. With "phenotype" is meant the complete physical, biochemical and physiological constitution of an orgm as determined in both genetic and environmental form and any other or any other group of such characteristics. In a preferred embodiment, the 5 'and / or 3' portions of the hairpin region of the HPH molecule are, independently, complementary to the target sequence. More specifically, the portion N and / or N 'of (N »N') 0 in the formulas I-VII are, in a manner independent, complementary to the objective sequence. In a preferred embodiment, the 5 'and / or 3' portions of the hairpin region of the HPH molecule are, independently, complementary to the target sequence. More specifically, the portions N and / or N 'of (F * F') h in formulas I-VI I are, independently, complementary to the target sequence. In a preferred embodiment, the invention features a method for modulating the function of an objective sequence that includes the steps of contacting the target sequence with the HPH molecules, including the molecules of the formulas l-XXV, under conditions appropriate for modulation. of the function of the target sequence. Such modulation can be carried out in vitro or in vivo, in microbial, plant or mammalian systems in which the modulation of the function could include the inhibition of the expression of the gene, the modification of the cellular function, change in the phenotype of the orgm , inhibition of the replication of a virus and / or viral RNA, inhibition of motility, migration of a cell and others. By "patient" is meant an orgm that is a donor or recipient of excised cells or of the cells themselves. "Patient" also refers to an orgm to which enzymatic nucleic acid molecules can be administered. Preferably, a patient is a mammal or mammalian cells. More preferred, a patient is a human or human cells. With "vectors" is meant any acid-based technique nucleic acid and / or virus used to deliver a desired nucleic acid. In another aspect, the nucleic acid molecule of the present invention is administered individually or in combination or in conjunction with other drugs, and can be used to treat diseases or conditions. For example, to treat a disease or condition associated with cancer, the patient could be treated, or other appropriate cells could be treated, as will be apparent to those skilled in the art. By "comprising" is meant to include, but is not limited to, anything that comes after the word "comprising". Therefore, the use of the term "comprising" indicates that the elements listed are required or mandatory, but that other elements are optional and may not be present. With "consisting of" it means that it is included, and that it is limited to, anything that comes after the phrase "consisting of". Thus, the phrase "consisting of" indicates that the items listed are required or mandatory, and other elements may not be present. With "consisting essentially of" means that any of the elements listed after the phrase is included, and is limited to other elements that do not interfere with or contribute to the activity or action specified in the description of the items listed. Therefore, the phrase "consisting essentially of" indicates that the items listed are required or mandatory, but that other elements are optional and may or may not be present depending on whether or not they affect the activity or action of the items listed.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.

DESCRIPTION OF THE PREFERRED MODALITIES First, the drawings will be briefly described.

Drawings Figure 1 is a schematic representation of the binding of the hairpin hybridizing molecule (HPH) to a target RNA molecule. During binding, both the 5 'sequence and the 3' sequence of the hairpin region may not be complementary to the target sequence. Alternatively, any of the 5 'or 3' sequences could be complementary to the objective RNA molecule independently. Figure 2A shows the hairpin structure of the unbound HPH nucleic acid molecule including a stem of four base pairs and an internal DNA sequence of nine nucleotides. The figure also shows the structure of the nucleic acid molecule before and after binding to RNA. These 5 'and 3' sequences of the molecule form the hairpin structure but not the base pairs with the target RNA molecule. Figure 2B shows the hairpin structure of the unbound nucleic acid molecule which also includes a stem of four base pairs and one DNA sequence of nine nucleotides. These 5 'and 3' sequences of the molecule form the hairpin structure. In some embodiments, the 5 'sequences and / or the 3' sequence can bind to the target RNA molecule independently. Figure 3 shows non-limiting structures of the molecules HPH that are within the scope of the present invention. In Figure 3A, (1) represents a circular nucleic acid molecule with an internal structure of hairpin stem with paired bases, each loop within the molecule comprises an activation region of RNase H and a non-activation region of RNase H and can join the target sequence; (2) represents a molecule comprising an RNase H activation region and an RNase H non-activating region that can interact with the target sequence and includes a hairpin region with internal paired bases and the additional nucleotide sequences at the 5 'and 3' ends of the hairpin structure which have equal or different lengths that could optionally be attached to the target sequences; (3) represents a molecule comprising an activation region of RNase H and a non-activation region of RNase H that can interact with the target sequence and a hairpin region with paired bases and the additional nucleotide sequence at the end 5 'of the hairpin stem structure which could optionally be attached to the target sequence; (4) represents a molecule comprising an activation region of RNAse H and a non-activation region of RNAse H that can interact with the sequence target and a hairpin region with internal paired bases and additional nucleotide sequences at the 3'-end of the hairpin structure that could optionally be bound in the target sequence; (5) represents a molecule comprising an RNase H activation region and an RNase H non-activating region that can interact with the target sequence and an internal hairpin stem region and an additional nucleotide sequence at the 5 'end and 3 'of the hairpin structure which are asymmetric in length that could optionally be joined in the target sequence; (6) represents a discontinuous circular nucleic acid molecule comprising an RNase H activation region and an RNase H non-activating region that can interact with the target sequence and an internal hairpin stem structure, each loop at the ends 3 'and 5' of the hairpin region can be linked independently to the target sequence. In Figure 3B, (1) represents an HPH nucleic acid molecule structure, in which the RNAse H activation region is at the 5 'end of the molecule and a portion of the RNAse H activation region forms a fork stem structure with a portion of the 3 'region of the HPH molecule. Both the RNase H activation region and the RNase H non-activation region can, independently or in combination, interact with the target sequence in a sequence-specific manner.; (2) and (5) represent a structure of HPH nucleic acid molecule, in which a portion of the RNAse H activation region forms a hairpin stem structure with a portion of the non-RNAse H activation region of the HPH molecule. Both the RNase H activation region and the RNase H non-activating region can, independently or in combination, interact with the target sequence in a sequence-specific manner; (3) and (6) represent a structure of HPH nucleic acid molecule, in which the portion of the RNase H activation region and a portion of the RNase H non-activating region form a hairpin stem structure with a portion of the non-RNAse H activation region located in a different part of the HPH molecule. Both the RNase H activation region and the RNase H non-activating region can, independently or in combination, interact with the target sequence in a sequence-specific manner; (4) represents a structure of HPH nucleic acid molecule, in which the RNase H activation region is at the 3 'end of the molecule and a portion of the RNase H activation region forms a stem structure of hairpin with a portion of the 5 'region of the HPH molecule. Both the RNAse H activation region and the non-activation region of RNase H can independently or in combination interact with the target sequence in a sequence-specific manner. In Figure 3C, (1) and (3) represent a structure of HPH nucleic acid molecule, in which a portion of the RNAse H activation region forms a hairpin stem structure with a portion of the region of RNase H activation of the HPH molecule. Both the RNAse H activation region and the non-activation regions of RNase H can, independently or in combination, interact with the target sequence in a sequence-specific manner, (2) and (4) represent a structure of HPH nucleic acid molecule, in which a portion of the region of RNAse H activation and a portion of the non-activation region of RNase H form a hairpin stem structure with a portion of the non-activation region of RNase H located in a different part of the HPH molecule. Both the RNAse H activation region and the non-activation regions of RNAse H can, independently or in combination, interact with the target sequence in a sequence-specific manner. Figure 4 shows a graph showing the effect of a mere HPH nucleic acid molecule of the present invention on the reduction of RNAse H levels of c-raf in PC-3 cells compared to controls without treatment and no matings . Cells were treated with nucleic acid molecules for 1, 3, or 5 days and then harvested to quantitate c-raf RNA. Figure 5 shows a graph showing the effect of a 33mer nucleic acid molecule of the present invention on the reduction of c-raf mRNA levels in PC-3 cells compared to untreated and unpaired controls. The cells were treated with the HPH nucleic acid molecules for 1, 3 or 5 days and then harvested to quantify the c-raf RNA. Figure 6 shows a graph showing the effect of a HPH 35mer nucleic acid molecule of the present invention on the reduction of c-raf mRNA levels in PC-3 cells compared to untreated and unpaired controls. The cells were treated with the nucleic acid molecules for 1, 3 or 5 days and then harvested to quantify c-raf RNA. Figure 7 shows a graph showing the effect of a simple HPH 31 nucleic acid molecule of the present invention on the reduction of c-raf mRNA levels in PC-3 cells compared to a non-pairing control. Figure 8 shows a graph showing the effect of a linear HPH 31 antisense molecule on the reduction of c-raf mRNA levels in PC-3 cells compared to a non-pairing control. Figure 9 shows the specific inhibition based on HPH nucleic acid molecule of c-raf RNA levels in PC-3 cells and the effect of, 2 and 4 do not base matings on this inhibition. Figure 10 shows several non-limiting examples of pseudonymous hairpin hybridizing molecules. Figure 10A is a pseudonymous hairpin hybridizing molecule consisting of two hairpin structures, and the target binding sequence located in close proximity to the 5 'end of the nucleic acid molecule compared to the 3' end. Figure 10B is a pseudonudo hairpin hybridizing molecule comprised of two hairpin structures, and a bone binding sequence. target located in close proximity to the 3 'end of the nucleic acid molecule compared to the 5' end. Figure 10C is a pseudonudo hairpin hybridizing molecule consisting of two hairpin structures, and two target binding sequences. Figure 10D is a pseudonymous hairpin hybridizing molecule consisting of two hairpin structures, a target binding sequence located in close proximity to the 3 'end of the nucleic acid molecule compared to the 5' end, and the sequences of additional nucleotides attached to the 5 'end and the 3' end of the hairpin hybridizing molecule. These additional sequences could be of equal or different length. Figure 10E is a pseudonymous hairpin hybridizing molecule consisting of two hairpin structures, two target binding sequences, and the additional nucleotide sequence linked at the 5 'and 3' ends of the hairpin hybridizing molecule. These additional sequences could be of equal length or of different length. Figure 10F is a pseudonymous hairpin hybridizing molecule consisting of two hairpin structures, a target binding sequence located in close proximity to the 5 'end of the nucleic acid molecule compared to the 3' end, and a sequence of additional nucleotide attached at the 5 'position of the hairpin hybridizing molecule. Figure 10G is a pseudonymous hairpin hybridizing molecule consisting of two hairpin structures, a target binding sequence located in close proximity to the 5 'end of the nucleic acid molecule compared to the 3' end, and additional nucleotide sequences linked at the 5 'and 3' ends of the hairpin hybridizing molecule. These additional sequences could be of the same length or of different length. Figure 11 shows a graph showing the effect of the HPH nucleic acid molecule of various configurations of the present invention on the reduction of c-raf mRNA levels in PC-3 cells compared to controls. Figure 12 shows a graph showing the effect of an HPH nucleic acid molecule of varying sizes and configurations of the present invention on the reduction of IMPDH II mRNA levels in PC-3 cells compared to untreated controls and unpaired Figure 13 shows a graph showing the effect of an HPH nucleic acid molecule of varying sizes and configurations of the present invention on the reduction of IMPDH II mRNA levels in PC-3 cells at an oligonucleotide concentration of 100 nM .

Mechanisms of action of the HPH nucleic acid molecules of the invention Antisense molecules known in the art are usually RNA or DNA oligonucleotides and function mainly by binding in a specific manner to the complementary sequences (mating) which results in the inhibition of synthesis of Peptides (Wu-Pong, Nov 1994, Bio Pharm, 20-33). The oligonucleotide binds to the target RNA by Watson Crick type base pairing and blocks gene expression by preventing ribosomal translation of the linked sequences either by spherical blocking of the RNAse H-mediated degradation of the target RNA. Antisense molecules could also alter protein synthesis by interfering with the processing or transport of RNA from the nucleus to the cytoplasm (Mukhopadhyay &Roth, 1996, Crit. Rev. In Oncogenesis 7, 151-190). In addition, the binding of a single chain DNA or RNA could result in heteroduplex nuclease degradation (Wu-Pong, supra; Crooke, supra). To date, the only modified base structure DNA chemistry that will function as substrates for RNase H are phosphorothioates and phosphorodithioates. It has recently been reported that oligonucleotides containing 2'-arabino and 2'-fluoroarabino- can also activate RNase H activity. A number of antisense molecules have been described using novel configurations of chemically modified nucleotides, secondary structure, and / or RNAse H substrate domains (Woolf et al., International PCT Publication No. Wo 98/13526; Thompson et al., USSN 60 / 082,404 which was filed on April 20, 1998, both of which are incorporated herein by reference in their entirety). The antisense molecules described in the art are essentially linear single-stranded oligonucleotides which are known to tolerate a number of non-matings and yet form stable hybrids with a target sequence raising concern about safety and toxicity in organisms. Although these molecules are functional, for some applications, including pharmaceutical compositions, greater specificity, lower toxicity and greater stability are desirable. The specificity of the oligonucleotides described above can be increased by using the HPH nucleic acid molecule of the present invention which forms internal hairpin structures with hydrogen bonding interactions. The partial complementarity of these HPH oligonucleotides with the target sequences does not allow an efficient opening of the internal hairpin structure of the HPH oligonucleotide resulting in a competition between a hairpin structure and the binding to target a sequence. Therefore, the hybridization interaction between HPH and the target sequences with one or more unpaired sequences is presented inefficiently, which makes them ineffective inhibitors of gene expression (see Figure 9). Tyagi & Kramer, 1996, Nat Biotechonol 14.303-308; Tyagi & Kramer, 1998, Nat Biotechnol 16, 359-363 have shown that oligonucleotides such as molecular markers having internal hairpin stems of 10 base pairs or more can bind to an objective sequence in a manner quite specific to the sequence in solution. . The specific interaction of a hairpin DNA with the target RNA has also been demonstrated in cells (Kostrikis et al., 1998, Science 279, 1228-1229) in which the Hairpin DNA to detect the presence of bFGF RNA, however, these oligonucleotides were not used to inhibit gene expression. In addition to increased specificity, the intramolecular binding of hairpin hybridizing molecules can result in increased stability. The hairpin sequences located at the respective ends of the oligonucleotide could increase the stability of these reagents because the lack of unpaired free nucleotides reduces the potential for degradation by exonucleases. The stabilization of the extremes is currently conferred by chemical modifications (phosphorothioate bonds etc.) which by themselves decrease the specificity, and possibly increase the cytotoxicity. The increased stability of ribozyme constructs based on hairpin-end vector has been demonstrated previously (Thompson et al., 1995, Nucleic Acids Research 23, 2259-2268). The effectiveness of these HPH molecules could be improved by the addition of nucleotides that act as substrates for RNase H within the molecule. However, the binding of DNA to RNA is not as thermodynamically as favorable as an RNA to RNA interaction (Altmann et al., 1996, Chlmia 50, 168-176). Therefore a molecule with both RNA and DNA nucleotides could have the ability to efficiently bind as well as promote the degradation of the RNA molecule by RNAse H. Inoe & Ohtsuka, 1987, Nucleic Acids Research 115, 6131, first proposed an oligonucleotide with a central region consisting of oligodeoxynucleotides flanked by nucleotide regions modified with 2'-0-methyl. The region of oligodeoxynucleotides in such chimeric molecules is recognized by RNase H when it binds to the target RNA; and facilitates the cleavage of the target RNA by RNase H. (Inoe &Ohtsuka, 1987, FEBS Lett 215, 327, Shibahara &Morivasa, 1987, Nucleic Acids Res. 15, 4403). It was proposed that these chimeric oligonucleotides interact with the target RNA more stably than an oligonucleotide consisting entirely of DNA. Alternatively, the nucleic acid molecule could function by binding to the target molecule which results in the spherical hindrance for ribosomal translation. A number of chemical modifications could be used with that strategy including the insertion of 2'-O-methyl modification in each nucleotide in the molecule. One of the most studied and used chemical alterations in oligonucleotides has been the modifications of base structure such as phosphorothioate, phosphorothioate, phosphorodithioate, 5'-thiosphate. Oligonucleotides of the phosphorothioate type are nucleic acid molecules whose phosphodiester bond has been modified by replacing a sulfur atom instead of an oxygen atom. In addition to increased resistance to the nuclease, oligonucleotides of the phosphorothioate, phosphorodithioate and 5'-thiosphosphate type are substrates for ribonuclease H (RNase H) (Monia, supra).; Crooke et al., 1995, Biochem. J. 3112, 599-608). RNase H is an endonuclease that catalyzes the degradation of RNA in a RNA-DNA heteroduplex (Hostomsky et al., 1993 in Nucleases, Linn et al., Eds., Cold Spring Harbor Laboratory Press, NY, 341-376). RNA / DNA heteroduplexes, called Okazaki fragments, are formed naturally during DNA replication. Therefore, the normal function of RNase H is to degrade the RNA portion of the heteroduplex to complete DNA replication. In experiments with E. coli RNAse H, the phosphorothioate type oligonucleotide activated the enzyme more efficiently (2-5 times) compared to an oligonucleotide containing standard phosphodiester (Crooke, 1995, supra), Synthesis of nucleic acid molecules The synthesis of nucleic acids with more than 100 nucleotides in length is difficult using automated methods, and the therapeutic cost of such molecules is prohibitive. In this invention, small nucleic acid motifs are used ("small" refers to nucleic acid motifs of no more than 100 nucleotides in length, preferably no more than 80 nucleotides in length and more preferred no more than 40 nucleotides in length for example the HPH nucleic acid molecules) for the exogenous delivery. The simple structure of these molecules increases the ability of the nucleic acid to invade selected regions as a target of the RNA structure. The oligodeoxynucleotide molecules of the present invention were chemically synthesized using standard protocols such as described in Caruthers et al., 1992, Methods in Enzymology 211, 3-19, which is incorporated in the present invention for reference. The synthesis method used for normal RNA follows the procedure as described in Usman et al., 1987 J. Am. Chem. Soc., 109, 7845; Scaringe et al 1995 Nucleic Acids Res. 23,2677-2684; Wincott et al., 1997, Methods Mol. Bio., 74, 59) and utilizes common nucleic acid protection and coupling groups such as dimethoxytrityl at the 5 'end and phosphoramidites at the 3' end and can be readily used to synthesize oligonucleotides containing 2'-O-alkyl. In a non-limiting example, small-scale synthesis was conducted on a synthesizer 394 from Applied Biosystems, Inc, using a modified protocol at 2.5 μmol with a 5 minute coupling step for the alkylsilyl-protected nucleotides and a coupling step of 2.5 minutes for 2'-0-methylated nucleotides. Table I indicates the quantities, and contact times, of the reagents used in the synthesis cycle. Alternatively, synthesis at 0.2 μmol scale can be performed on a 96-well plate synthesizer such as the instrument produced by Protogene (Palo Alto, CA) with the minimum modification to the cycle. A 15-fold excess (31 μl of 0.1 M = 3.1 μmol) of phosphoramidite and a 38.7-fold excess of S-ethyl tetrazole (31 μl of 0.25 M = 7.75 μmol) relative to 5'-hydroxyl bound to polymer were used. in each cycle of copulation. The average coupling yields in synthesizer 394 of Applied Biosystems, Inc. determined by colorimetric quantitation of trityl fractions, were 97.5-99%. The other reagents for oligonucleotide synthesis used with synthesizer 394 from Applied Biosystems, Inc., included solution for detritylation with 3% TCA in methylene chloride (ABI); blocking ends was performed with 16% N-methyl imidazole in THF (ABI) and 10% acetic anhydride / 10% 2,6-lutidine in THF (ABI); the oxidation solution was 16.9 mM of l2, 49 mM of pyridine, 9% of water in THF (PERSEPTIVE ™). Grade synthesis acetonitrile from Burdick & Jackson, directly from the reagent bottle, the solution of S-ethyltetrazole (0.25 M in acetonitrile) was prepared from the solid obtained from American International Chemicals, Inc. The deprotection of the oligonucleotides of the present invention was performed using a protocol of two containers or a single container protocol. For the two vessel protocol, the trityl-on oligonucleotide bound to polymer was transferred to a glass jar with a screw cap of 4 ml and suspended in an aqueous 4% methylamine solution (1 ml) at 65 ° C for 10 minutes. minutes After cooling down to -20 ° C, the supernatant was removed from the polymeric support. The support was washed three times with 1 ml of EtOH: MeCN: H2? / 3: 1: 1, subjected to swirling and then the supernatant was added to the first supernatant. The combined supernatants, containing the oligoribonucleotide, were dried to a white powder. The base-deprotected oligonucleotide was resuspended in anhydrous TEA / HF / NMP solution (300 μl of a 1.5 ml solution of N-methylpyrrolidone, 750 μl of TEA and 1 ml of TEA «3HF to provide a 1.4 M HF concentration ) and heated to 65 ° C. After 1.5 hours, the oligomer was quenched with NH HC31.5.5 M. Alternatively, for the single container protocol, the trityl-on oligonucleotide bound to polymer was transferred to a glass jar with a screw cap of 4 ml and suspended in a solution of methylamine and suspended in a solution of 33% methylamine / DMSO: 1/1 (0.8 ml). 65 ° C for 15 minutes. The glass bottle was brought to room temperature. TEA »3HF (0.1 ml) was added and the glass flask was heated at 65 ° C for 15 minutes. The sample was cooled to -20 ° C and then quenched with NH 1.5 3 1.5 M. For the purification of the oligonucleotides with trifly, the solution quenched with NH HC? 3 was loaded onto a cartridge containing previously washed C-18. with acetonitrile followed by 50 nM TEAA. After washing the cartridge loaded with water, the RNA was detritylated with 0.5% TFA for 13 minutes. The cartridge was then washed again with water, salt exchanged with 1 M NaCI and washed again with water. The oligonucleotides were then eluted with 30% acetonitrile. Alternatively, or in addition to, the methods described in the present invention, the oligonucleotides of the present invention can be purified by other methods known in the art, for example, see Sproat et al., 1999, Nucleic Acids Res., 27 , 1950). The average step coupling yields were > 98% (WIncott et al., 1995, Nucleic Acids Res., 23, 2677-2684). Those skilled in the art will recognize that the synthesis scale can be adapted to be larger or smaller than that of the example described above including, but not limited to, a 96 cavity format, all that matter is the ratio of chemical compounds used in the reaction. Alternatively, the nucleic acid molecules of the present invention can be synthesized separately and can be pooled after synthesis, for example by ligation (Moore et al., 1992, Science 256, 9923; Draper et al., Pub. PCT International No. WO 93/23569, Shabarova et al., 1991 Nucleic Acids Res., 19, 4247, Bellon et al., 1997, Nucleosides &Nucleotides, 16, 951, Bellon et al., 1997, Bioconjugate Chem 8,204).

Administration of Nucleic Acid Molecules Methods for delivering nucleic acid molecules are described in Akhtar et al., 1992, Trends Cell Bio., 2, 139; and in Delivery Strategies for Antisense Ollgonucleotide Therapeutics, Ed. Akhtar, 1995, of which both are incorporated for reference in the present invention, Sullivan et al., PCT WO 94/02595, also describe the general methods for the delivery of Enzymatic RNA These protocols could be used for the delivery of virtually any nucleic acid molecule. The nucleic acid molecules could be administered to the cells by a variety of methods known to those skilled in the art, including, but not limited to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins , biodegradable nanocapsules and bioadhesive microspheres. For some indications, nucleic acid molecules they could be delivered directly ex vivo to the cells or tissues with or without the aforementioned vehicles. Alternatively, the nucleic acid / carrier combination is delivered locally by direct injection or through the use of a catheter, infusion pump or stent. Other routes of administration include, but are not limited to, delivery by intravascular, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral, (in tablet or pill form), topical, systemic, ocular, intraperitoneal and / or intrathecal In Sullivan et al., Supra and in Draper et al., PCT WO 93/23569, more detailed descriptions are provided about the delivery and administration of nucleic acid, both documents being incorporated for reference in the present invention. The molecules of the present invention can be used as pharmaceutical agents. Pharmaceutical agents prevent, inhibit the appnce, or treat (alleviate a symptom to some degree, preferably all symptoms) of a pathological condition in a patient. Polynucleotides of the invention, negatively charged, can be administered (eg, RNA, DNA or protein) and introduced to a patient by any of the standard means, with or without stabilizers, regulatory solutions and the like, to form a pharmaceutical composition. When it is desired to use a liposome delivery mechanism, standard protocols for the formation of the iiposomes can be followed. The compositions of the present invention could also be formulated and used as tablets, capsules, or elixirs for oral administration; suppositories for rectal administration, sterile solutions and suspensions for administration by injection; and similar. The present invention also includes pharmaceutically acceptable formulations of the disclosed compounds. The formulations include the salts of the above compounds, for example acid addition salts, for example the salts of the hydrochloric, hydrobromic, acetic and benzene-sulfonic acids. A "pharmaceutical composition" or "pharmaceutical formulation" refers to a composition or formulation in a form suitable for administration, eg, systemic administration, in a cell or patient, preferably a human. The appropriate forms depend, in part, on the use or the route of entry, for example, orally, transdermally, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which it is desired to deliver the negatively charged polymer). For example, pharmacological compositions injected into the bloodstream must be soluble. Other factors are known in the art, and include considerations such as toxicity and ways that prevent the composition or formulation from exerting its effect. By "systemic administration" is meant the absorption or systemic accumulation in vivo of drugs that are found in the bloodstream followed by distribution throughout the body. The routes of administration that lead to systemic absorption include, without limitations, the intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular routes. Each of these routes of administration exposes the desired, negatively charged polymers, eg, nucleic acids, to an accessible diseased tissue. It has been shown that the rate of entry of a drug into the bloodstream is a function of weight or molecular size. The use of a liposome or other drug vehicle comprising the compounds of the present invention can potentially locate the drug, for example, in certain tissue types, such as the tissues of the Endothelial Reticular System (SER). A liposome formulation that can facilitate the association of the drug with the surface of the cells, such as lymphocytes and macrophages, is also useful. This method could provide improved delivery of the drug to target cells by taking advantage of the specificity of macrophages and lymphocytes in immune recognition of abnormal cells, such as cancer cells. The invention also relates to the use of the composition comprising liposomes with modified surface containing lipids with polyethylene glycol (liposomes modified with PEG or long circulating liposomes or furtive liposomes). These formulations offer a method to increase the accumulation of drugs in the target tissues. This vehicle class for the drug resists opsonization and elimination mediated by the mononuclear phagocyte system (MPS or RES), thus allowing larger times of circulation in the bloodstream and times of death. improved exposures for the encapsulated drug (Lasic et al., Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull., 1995, 43, 1005-1011). It has been shown that such liposomes accumulate selectively in tumors, probably by extravasation and capture in neovascularized target tissues (Lasic et al., Science 1995, 267 1275-1276, Oku et al., 1995, Biochim. Biophys. , 1238, 86-90). Liposomes with prolonged circulation improve the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared with conventional cationic liposomes which are known to accumulate in MPS tissues (Liu et al., J. Biol. Chem. 1995, 42, 24864-24870; Choi et al., PCT International Publication No. WO 96/10391; Ansell et al., PCT International Publication No. WO 96/10390; Holland et al., PCT International Publication No. WO 96/10392; of which all are incorporated for reference in the present invention). It is also likely that prolonged circulation liposomes protect drugs from nuclease-mediated degradation to a greater degree compared to cationic liposomes, based on their ability to prevent accumulation in metabolically aggressive MPS tissues such as liver and blood. spleen. The present invention also includes compositions prepared for storage or administration which include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. The vehicles or diluents for therapeutic use are well known in the pharmaceutical area, and are describe for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro editors 1985) incorporated in the present invention for reference. For example, preservatives, stabilizers, colorants and flavorings could be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition, antioxidants and suspending agents could be used. A pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence or treat (alleviate a symptom to some degree, preferably all symptoms) of a pathological condition. The pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, the concurrent drugs and other factors that will be recognized by the animals. experts in the medical technique. In general, an amount between 0.1 mg / kg and 100 mg / kg of body weight / day of active ingredients is administered depending on the potency of the negatively charged polymer. The nucleic acid molecules of the present invention could also be administered to a patient in combination with other therapeutic compounds to increase the overall therapeutic effect. The use of multiple compounds to treat an indication could increase the beneficial effects and reduce the presence of side effects at the same time.

Alternatively, some of the nucleic acid molecules of the present invention (for example those of formula IV) can be expressed within the cells from eukaryotic promoters (see for example, Izant and Weintraub, 1985 Science 229, 345; McGarry and Lindquist, 1986 Proc. Natl. Acad. Sci. USA 83, 399; Scanlon et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992 , Antisense Res. Dev., 2, 3-15; Dropulic et al., 1992, J. Virol., 66, 1432-41; Weerasinghe et al., 1991, J. Virol, 65, 5531-4; Ojwang et al. al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Sarver et al., 1990, Science 247, 1222 -1225, Thompson et al., 1995, Nucleic Acids Res., 23, 2259, Good et al., 1997, Gene Therapy, 4, 45, all of which are incorporated by reference in the present invention in their entirety). Those skilled in the art will recognize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA / RNA vector. The activity of such nucleic acids can be increased by releasing them from the primary transcript by a ribozyme (Draper et al., PCT WO 93/23569, and Sullivan et al., PCT WO 94/02595, Ohkawa et al., 1992 Nucleic Acids Symp. Ser., 27, 15-6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura et al., 1993, Nucleic Acids Res., 21, 3249-55; Chowira et al., 1994, J. Biol. Chem. 269, 25856; all of these references, in their entirety, are incorporated in the present invention for reference). In another aspect of the invention, the RNA molecules of the present invention are preferably expressed from the units of transcript (see for example Couture et al., 1996, TIC, 12, 510) inserted into DNA or RNA vectors. Recombinant vectors are preferably DNA plasmids or viral vectors. Viral vectors expressing ribozyme can be constructed based on, but not limited to, adeno-associated viruses, retroviruses, adenoviruses, or alphaviruses. Preferably, recombinant vectors that can express the nucleic acid molecules are delivered as described above, and persist in the target cells. Alternatively, viral vectors that ensure transient expression of the nucleic acid molecules could be used. Such vectors could be administered repeatedly as needed. Once expressed, the nucleic acid molecule binds to the target mRNA. The supply of vectors that express the nucleic acid molecule could be systemic, for example, by intravenous or intramuscular administration, by administration to the target cells excised from the patient followed by reintroduction of the same to the patient, or by any other means that allows introduction into the desired target cell (for a review see Couture et al., 1996, TIG., 12, 510). In one aspect the invention features the description of an expression vector comprising the nucleic acid sequence encoding at least one of the nucleic acid molecules of the present invention. The nucleic acid sequence encoding the nucleic acid molecule of the present invention is operably linked in a form that allows expression of that nucleic acid molecule.

In another aspect the invention features an expression vector comprising: a) a transcription initiation region (e.g., the eukaryotic pol I, II or III initiation region); b) a transcription termination region (e.g., the eukaryotic pol I, II or 111 terminator region); c) a gene encoding at least one of the nucleic acid catalysts of the present invention; and wherein said gene is operably linked to said start region and said termination region in a manner that allows expression and / or delivery of said nucleic acid molecule. Optionally, the vector could include an open reading frame (ORF) for a protein that is operably linked on the 5 'side or on the 3' side of the gene encoding the nucleic acid catalyst of the invention; and / or an intron (intermediate sequences). Transcripts of the sequences of the nucleic acid molecules are driven from a promoter for RNA polymerase I (pol I), eukaryotic RNA polymerase II (pol II) or eukaryotic lll (pol lll) RNA polymerase. Transcripts from the promoters for pol II or pol III will be expressed at high levels in the cells; the levels of a given pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (promoters, silencers, etc.) that are present in the surroundings. Prokaryotic RNA polymerase promoters are also used, provided that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gao and Huang, 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993, Methods Enzymol., 217, 47-66; Zhou et al., 1993, Mol. Cell. Biol., 10, 4529-37). Several investigators have demonstrated that nucleic acid molecules, such as ribozymes expressed from such promoters, can function in mammalian cells (see for example, Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 6340-4, L'Huiller et al., 1992, EMBO J "11, 4411-8, Lisziewics et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 8000-4, Thompson et al., 1995, Nucleic Acids Res., 23, 2259, Sullenger &Cech, 1993, Science, 262, 1566). More specifically, transcription units such as those derived from genes encoding small nuclear U6 (snRNA), transfer RNA (tRNA) and adenovirus VA RNA, are useful for generating high concentrations of molecules of desired RNAs such as ribozymes in cells (Thompson et al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al., 1994, Nucleic Acids Res., 22, 2830; Noonberg et al., US patent No. 5,624,803, Good et al., 1997, Gene Ther.4,45, Beigeiman et al., PCT International Publication No. WO 96/18736, all of which are incorporated by reference in the present invention). The above ribozyme transcription units can be incorporated into a variety of vectors to be introduced into mammalian cells, including but not restricted to, plasmid vectors of DNA, viral DNA vectors, (such as vectors of adenovirus or adeno-associated virus) or viral RNA vectors (such as retroviral vectors or alphaviruses) (for a review see Couture and Stinchcomb, 1996, supra). In yet another aspect, the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the nucleic acid molecules of the invention, in a form that allows the expression of that nucleic acid molecule. The expression vector comprises in one embodiment: a) a transcription initiation region b) a transcription termination region; c) a gene encoding said at least one nucleic acid molecule; and wherein said gene is operably linked to said start region and said termination region in a manner that allows expression and / or delivery of said nucleic acid molecule. In another preferred embodiment, the expression vector comprises: a) a transcription initiation region b) a transcription termination region; c) an open reading frame; d) a gene encoding said at least one nucleic acid molecule; wherein said gene is operably linked to the 3 'end of said open reading frame, and wherein said gene is operably linked to said start region, said open reading frame and said termination region in a form that allows the expression and / or delivery of said nucleic acid molecule. Even in another embodiment, the expression vector comprises: a) a transcription initiation region b) a transcription termination region transcription; c) an intron; d) a gene encoding said at least one nucleic acid molecule; and wherein said gene is operably linked to said start region, said intron, and said termination region in a form that permits expression and / or delivery of said nucleic acid molecule. In a further embodiment, the expression vector comprises: a) a transcription initiation region b) a transcription termination region; c) an intron; d) an open reading frame; e) a gene encoding said at least one nucleic acid molecule; wherein said gene is operably linked to the 3 'end of said open reading frame; and wherein said gene is operably linked to said start region, said intron, said open reading frame and said termination region in a manner that allows the expression and / or delivery of said nucleic acid molecule.

Optimizing the activity of the nucleic acid molecule Chemically synthesizing the nucleic acid molecules with modifications (base, sugar and / or phosphate) that prevent their degradation by serum ribonucleases could increase their potency (see for example, Eckstein et al. , PCT International Publication No. WO 92/07065, Perrault et al., 1990, Nature, 344, 565, Pieken et al., 1991, Science, 253, 314, Usman and Cedergren, 1992, Trends in Biochem. 17, 334; Usman et al., PCT International Publication No. WO 93/15187; and Rossi et al., PCT International Publication No. WO 91/13162; Sproat, patent E.U.A. No. 5,334,711; and Burgin et al., supra; all these documents describe various chemical modifications that can be made to the base, phosphate, and / or sugar portions of the nucleic acid molecule of the present invention). Modifications are desired that improve their efficiency in the cells, and the elimination of bases of the nucleic acid molecules to shorten the times of oligonucleotide synthesis and reduce the chemical requirements. (All these publications are therefore incorporated for reference in the present invention). There are several examples in the art that describe modifications to bases, sugar and phosphate that can be introduced into nucleic acid molecules with significant improvements in their nuclease stability and efficacy. For example, oligonucleotides are modified to improve their stability and / or improve their biological activity by modification with nuclease-resistant groups, for example, modifications to the base of the 2'-amino, 2'-fluoro, 2'-0 nucleotide -methyl, 2'-H, (for a review see Usman and Cedergren, 1992 TIBS, 17, 34; Usman et al., 1994 Nucleic Acids Symp. Ser. 31, 163; Burgin et al., Biochemistry 35, 14090) . Sugar modifications of nucleic acid molecules have been extensively described in the art (see Eckstein et al., PCT International Publication No. WO 92/07065, Perrault et al., 1990, Nature, 344, 565-568; Pieken et al., 1991, Science, 253, 314-317; Usman and Cedergren, 1992, Trends in Biochem. Sci., 17, 334-339; Usman et al., Publication PCT International No. WO 93/15187; Sproat, patent E.U.A. No. 5,334,711; Beigelman et al., 1995, J. Biol. Chem. 270, 25702; Beigelman et al., PCT International Publication No. WO 97/26270; Beigelman et al., Patent E.U.A. No. 5,716,824; Usman et al., Patent E.U.A. No. 5, 627,053; Woolf et al., PCT International Publication No. WO 98/13526; Thompson et al., USSN 60 / 082,404 which was filed on April 20, 1998; Karpeisky et al., 1998, Tetrahedron Lett. 39, 1131; of which all are incorporated in the present invention for reference in their entirety). Such publications, which describe general methods and strategies for determining the site of incorporation of sugar, base and / or phosphate modifications, and the like into ribozymes without inhibiting catalysis, are incorporated for reference in the present invention. In view of such teachings, similar modifications can be used as described in the present invention to modify the HPH molecules and the HPH molecules of the present invention. Although chemical modification of the internucleotide bonds of the oligonucleotide with phosphorothioate, phosphorothioate and / or 5'-methylphosphonate linkages improves stability, a very high amount of these modifications could cause increased toxicity. Therefore, when designing HPH molecules, the amount of these internucleotide bonds should be minimized. The reduction in the concentration of these bonds should reduce the toxicity resulting in an increased efficacy and a higher specificity of these HPH molecules.

Nucleic acid molecules having chemical modifications that maintain or improve activity are described in the present invention. Such a nucleic acid is also, generally, more resistant to the nuclease than the unmodified nucleic acid. Therefore, in a cell and / or in vivo, the activity may not be significantly reduced. The HPH therapeutic molecules delivered exogenously should, optimally, be stable within the cell until the translation of the target RNA has been sufficiently inhibited to reduce the levels of the unwanted protein. This period varies from several hours to days depending on the pathological state. Clearly, the nucleic acid molecules must be resistant to the nucleases to function as effective intracellular therapeutic agents. Improvements in chemical synthesis of RNA and DNA (WIncott et al., 1995, Nucleic Acids Res., 23, 2677; Caruthers et al., 1992, Methods in Enzymology 211, 3-19; incorporated in the present invention for reference ) have expanded the ability to modify the nucleic acid molecules by introducing nucleotide modifications to improve their stability to the nuclease as described above. The use of these HPH molecules could lead to a better treatment of disease progression, providing the possibility of combination therapies (multiple HPH molecules targeting different genes, HPH molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of HPH molecules (including different HPH motifs) and / or some other biological or chemical molecules). He Treatment of patients with nucleic acid molecules could also include combinations of different types of nucleic acid molecules.

Validation of the objective One of the most challenging tasks in the discovery of drugs is the choice of a therapeutic target. Historically, traditional biochemical studies and other studies have offered limited information in this regard. However, recent advances in genomics offer the potential to revolutionize both the speed and the certainty of the identification of the therapeutic goal. Progress in the characterization of genes in the human genome has been very rapid, and it is currently estimated that the sequence of the entire complement of genes in the human genome could be determined before the end of this century. However, this mass of information is reaching the scientific world without a map. Converting the pure information of the sequence of a gene into a functional understanding regarding its intervention in human disease has proven to be a much more difficult problem. Even after associating a group of genes with a particular disease, the process of validating which genes are appropriate to be used as therapeutic targets is often slow and costly. Currently, most companies engaged in genomic activities have access to a multitude of partial or complete sequences, but do not possess the appropriate technologies to determine which of those sequences is an appropriate therapeutic objective. As a result, only a few genes have been unequivocally identified as the causative agent of a specific disease. The nucleic acid molecules of the present invention can inhibit the expression of genes in a highly specific manner by binding to, and causing cutting of the mRNA corresponding to the gene of interest, and thereby preventing the production of the gene product (Christoffersen, Nature Biotech. , 1997, 2, 483-484). Appropriate delivery vehicles can be combined with these nucleic acid molecules (including polymers, cationic lipids, liposomes and the like) and delivered to appropriate cell cultures or in animal disease models in vivo as described above. By monitoring the inhibition of gene expression and correlation with phenotypic results, the relative importance of the particular gene sequence can be established with the pathology of the disease. The procedure can be both rapid and highly selective, and allows the procedure to be used at any point in the body's development. The novel chemical composition of these nucleic acid molecules could allow for aggregate stability and therefore increased efficiency.

EXAMPLES The following are non-limiting examples demonstrating the utility of the nucleic acid molecules of the present invention. The Those skilled in the art will recognize that some experimental conditions such as temperatures, reaction times, media conditions, reagents for transfection, cell types and tests for RNA are not intended to be easily modified without significantly altering the protocols.

EXAMPLE 1 Identification of potential binding sites for the HPH molecule in the target sequence The sequences of the target RNA molecules were examined relative to the accessible sites using a computer folding algorithm. The regions of the mRNA that did not form structures with secondary sticking were identified. A more elaborate protocol can be found to identify the appropriate objectives in Stinchcomb et al., Patent E.U.A. No. 5,646,042 which is incorporated herein by reference in its entirety.

EXAMPLE 2 Negative regulation of the expression of c-raf Using standard protocols, HPH oligonucleotides were selected to target exon 11 of the human c-raf gene, with 4-6 complementary nucleotides at the 5 'end and at the 3' end (see Wincott et al., Supra). These 5 'and 3' sequences were not complementary to the c-raf target. Of the 23 nucleotides complementary to the target sequence, 11 nucleotides were exchanged in the DNA center and in the RNA arms to generate a control molecule lacking the ability to negatively regulate the c-raf mRNA in a specific form to the sequence. The sequences for the nucleic acid molecules that were used are shown in Table III.

Tissue culture and nucleic acid delivery protocol Prostate cancer cells (PC-3) were cultured in a growth medium consisting of Kaighn's F-12K medium, 10% FBS, 1% glutamine, 20 mM HEPES, 1% penicillin / streptomycin up to sub-confluent densities. A 4X concentration (10 μg / ml) of GSV (Glen Research) was prepared from a stock solution of 2 mg / ml as well as a 10 μM solution of the nucleic acid molecule of the present invention and its antisense control. Antisense and GSV complexes were formed in a 96-well plate by pipetting the antisense and GSV channel to form complex solutions having twice the final concentrations.

Inhibition of c-raf mRNA using variable length nucleic acid molecules Using the cell culture and oligonucleotide delivery method described above, PC-3 cells were treated for 1, 3, or 5 days with hairpin oligonucleotides complexed with lipids . The oligonucleotides used are 31 nucleotides (SEQ ID No. 12022), 33 nucleotides (SEQ ID No. 12021), or 35 nucleotides (SEQ ID No. 12020) in length. Non-pairing controls were used to verify for non-specific effects and are given previously as SEQ. ID. Nos. 12023, 12024 and 12025 for 35, 33 and 31 numbers, respectively. An untreated control was also evaluated to determine the basal levels of c-raf. Then, PC-3 cells were harvested with 150 μl of RLT regulatory solution for lysis (Qiagen). The RNA was purified using the Qiagen instructions and the RNA was quantified using TaqMan ™ reagents (Perkin Elmer) and the 7700 Prism apparatus (Perkin Elmer) using the manufacturer's protocol. The ratio of c-raf mRNA to β-actin mRNA was determined by real-time PCR after reverse transcription. The results are shown in figures 3-5. The results show that all three molecules showed high levels of c-raf mRNA reduction compared to non-mating controls without taking into account the length of the oligonucleotides. Inhibition levels varied from 80 to 93% in PC-3 cells. After each designated period, PC-3 cells were harvested with 150 μl of RLT regulatory solution for lysis (Qiagen). The RNA was purified using the Qiagen instructions and the RNA was quantified using TaqMan ™ reagents (Perkin Elmer) and the 7700 Prism apparatus (Perkin Elmer) using the manufacturer's protocol. The ratio of c-raf mRNA to β-actin mRNA was determined by real-time PCR after reverse transcription.

EXAMPLE 3 Comparison of the inhibition of c-raf between hairpin hybridizing molecules and a linear antisense molecule To assess whether the nucleic acid molecules of the present invention could or could not inhibit c-raf mRNA as well as linear antisense molecules, hairpin molecules and linear antisense molecules were synthesized (Wincott et al., Supra). Using the cell culture and oligonucleotide delivery method described in Example 2, PC-3 cells were treated for 1, 3, or 5 days with hairpin oligonucleotides complexed with lipids or with a linear antisense molecule complexed with lipid. The hairpin molecule (SEQ ID No. 5) has a length of 31 nucleotides and the results of the inhibition of c-raf were compared with a non-pairing control (SEQ ID No. 6). The potency of the antisense molecule was also compared to its non-mating control. After each designated period, PC-3 cells were harvested with 150 μl of RLT regulatory solution for lysis (Qiagen). The RNA was purified using the Qiagen instructions and the RNA was quantified using TaqMan ™ reagents (Perkin Elmer) and the 7700 Prism apparatus (Perkin Elmer) using the manufacturer's protocol. The ratio of c-raf mRNA to β-actin mRNA was determined by real-time PCR after reverse transcription. The data are given in Figures 7 and 8. The HPH molecules significantly reduce the level of c-raf RNA while the non-pairing molecules do not cause any significant reduction (Figures 6, 7). Similarly, the linear antisense molecule reduced r-raf RNA levels significantly. These experiments demonstrate that the magnitude of the c-raf inhibition caused by a hairpin oligonucleotide can be compared to a linear molecule lacking the complementary 5 'and 3' ends of hairpin.

EXAMPLE 4 Mutation analysis of the hairpin hybridizing molecule The nucleic acid molecules of the present invention were designed to bind to the c-raf message (Table II) with 0, 1, 2, or 4 non-matings within the internal DNA sequence. The molecules were designed in such a way that the 5 'sequence is complementary both to the 3' sequence as well as to the target molecule. These molecules were delivered to PC-3 cells using the cell culture protocol and oligonucleotide delivery described in Example 2. The lipid / nucleic acid molecule complexes were added to the cells and allowed to associate for 24 hours. Then the PC-3 cells were harvested with 150 μl of RLT regulatory solution for lysis (Qiagen). The RNA was purified using the Qiagen instructions and the RNA was quantified using TaqMan ™ reagents (Perkin Elmer) and the 7700 Prism apparatus (Perkin Elmer) using the manufacturer's protocol. The ratio of c-raf mRNA to β-actin mRNA was determined by real-time PCR after reverse transcription. The results are shown in Figure 9. Only a single mutation within the HPH nucleic acid molecule is sufficient to destroy the inhibitory effects of the HPH molecule. This demonstrates that the HPH molecules of the present invention are highly sequence specific reagents.

EXAMPLE 5 Inhibition of the expression of IMDPH II RNA with hairpin hybridizing molecules of varying lengths Prostate cancer cells (PC3) were cultured as described above. The nucleic acids were complexed and applied to the cells as described, with the exception that a cationic lipid was used.

The final concentration of the oligonucleotide was 100nM. RNA levels were measured by TaqMan ™ analysis as described above.

Using the cell culture and oligonucleotide delivery method described above, PC3 cells were treated for 24 hours with oligonucleotides complexed with lipid. The oligonucleotides targeted to IMPDH II each had a target hybridization region 23mer plus a 6-base fork hybridization region at the 3 'end that are fixed to the 5' end (SEQ ID NO: 11 and SEQ. ID No. 12 of non-pairing control of 2 bases). Alternatively, the oligonucleotide had a 19mer target hybridization region plus a 4-base fork hybridization region at the 3 'end that is fixed to the 5' end (SEQ ID No. 13, and SEQ ID. No. 14 non-pairing control of 2 bases). As shown in Figure 12, the hairpin hybridizing molecule exhibited 45-70% inhibition of the target RNA level relative to untreated cells. In both cases, a non-pairing of 2 bases was sufficient to avoid negative regulation of the target, which demonstrates the high specificity of these reagents.

EXAMPLE 6 Alternative fork hardening domains that confer comparable efficiency in cell culture Using the cell culture and oligonucleotide delivery method described above, PC3 cells were treated for 24 hours with oligonucleotides complexed with lipid. The oligonucleotides targeted to exon 11 of c-Raf, and consisted of DNA center regions (an example of Rnasa activation region) at or near the 5 'end of the oligonucleotide, and a 3' hairpin hybridization region that was it can tune to different regions of the complementary region of the target, including the DNA center and / or the RNA arms. As shown in Figure 11, a region of hybridization to target-only 21 with a 6-base fork that is fixed to the 5 'end of the oligonucleotide that overlaps a portion of the DNA center (SEQ ID No. 15) exhibits a inhibition greater than 80% > of the expression of the target RNA. Mixing the 3 'end to avoid the formation of an intramolecular hairpin (SEQ ID No. 16) does not improve or interfere with the efficiency of the cell in this test, indicating that the oligonucleotide could present base pairing with Target RNA in equivalent form with or without the fork structure. A 18mer (complementary) target hybridization region with self-complementary structures of 6 variable nucleotides (SEQ ID Nos. 17, 18, 19, 20) exhibits 35-65% inhibition of target RNA expression, indicating that A variety of structures could result in effective molecules. The difference in magnitude between the 21 mer and 18mer structures probably reflects the lower target binding affinity of the 18mer structure. The differences in magnitude of inhibition between SEQ. ID. Nos. 17, 18, 19, 20 could reflect subtle variations of sequence dependence on optimal structure. Using the cell culture and delivery method Oligonucleotides described above, PC3 cells were treated for 24 hours with oligonucleotides complexed with lipid. The oligonucleotides targeted IMPDH II, and consisted of DNA center regions at the center of the oligonucleotide, and a 3 'hairpin hybridization region that can be annealed to the DNA center. As shown in Figure 13, a linear antisense oligonucleotide with a 23mer target hybridization region (SEQ ID No. 22) gave 70% inhibition, while negative controls of random sequence and altered sequence (SEQ. ID No. 21, 23) virtually no inhibition. The hairpin oligonucleotides (SEQ ID No. 24, 25) with either a 4 or 6 base hairpin that binds to the DNA center showed 60-70% inhibition of target RNA expression. In another example, a linear oligonucleotide that selects a different target site in the target RNA (SEQ ID No. 26) gave 80% inhibition, and the hairpin oligonucleotides (SEQ ID Nos. 27, 28) gave 70-80 % inhibition of target RNA expression. Those skilled in the art will recognize that the present invention is not limited to the HPH molecules used in the previous examples. The present invention broadly characterizes HPH oligonucleotides of varying structures, including those that hybridize to the internal RNase activation regions, those that hybridize to both the RNase activation region and the RNase non-activation region, and those that hybridize to the RNase activation region. hybridize to an RNAse activation region at either the 5 'or 3' end (see for example Figures 1-3 and 10).

The hairpin structure could provide protection against exonucleolytic and / or endonucleolytic degradation, thereby increasing stability both in vivo and in vitro. The fork creates a duplex region that juxtaposes various chemical modifications of the end that could confer pharmacokinetics or distribution in altered in vivo tissues. Therefore these molecules could have advantages compared to traditional linear antisense molecules for their use as therapeutic agents or as tools for the validation of the target in vivo.

Diagnostic Uses The nucleic acid molecules of the present invention could be used as diagnostic tools to examine genetic shifts and mutations within diseased cells or to detect the presence of specific RNA molecules in a cell. The close relationship ben the antisense activity and the structure of the target RNA allows the detection of mutations in any region of the molecule that alters the base pairing and the three-dimensional structure of the target RNA. By using multiple nucleic acid molecules described in this invention, one could map the nucleotide changes that are important for the RNA structure and its function in vitro, as well as in cells and tissues. The inhibition of the target RNA molecules with nucleic acid molecules could be used to inhibit the expression of the gene and define the (essentially) role of the products of the gene specified in the invention. advancement of the disease. In this way, other genetic targets could be defined as important mediators of the disease. These experiments will lead to better treatment during the progression of the disease by providing the possibility of combination therapies (eg, multiple nucleic acid molecules targeting different genes, nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of nucleic acid molecules and / or some other chemical products or biological molecules). Other in vitro uses of the nucleic acid molecules of this invention are known in the art, and include the detection and presence of RNA molecules related to various conditions. All patents and publications mentioned in the specification indicate the skill levels of those skilled in the art to which the invention pertains. All references cited in this description are incorporated for reference to the same extent as if each reference has been incorporated for reference in its entirety individually. One skilled in the art will readily appreciate that the present invention is well adapted to realize the objectives and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and compositions described in the present invention as currently representative of the preferred embodiments are by way of example and are not intended to be limitations on the scope of the invention. invention. Changes in the same and other uses will be apparent to those skilled in the art, which are encompassed within the essence of the invention, are defined by the scope of the claims. Variable substitutions and modifications may be made, which will be apparent to those skilled in the art, to the invention described herein without departing from the scope and scope of the invention. Therefore, such additional embodiments are within the scope of the present invention and the following claims. The invention described in an illustrative manner herein may be practiced in an appropriate manner in the absence of any element or elements, limitation or limitations that are not specifically described therein. Thus, for example, in each case in the present invention any of the terms "comprising", "consisting essentially of" and "consisting of" could be replaced with any of the other two terms. The terms and expressions that have been used are used as descriptive terms and not as limiting, and there is no intention that during the use of such terms and expressions is excluded any of the equivalents of the characteristics shown and described or portions thereof , but it is recognized that various modifications are possible within the field of the claimed invention. Therefore, it should be understood that while the present invention has been specifically described by preferred embodiments, those skilled in the art may resort to the features, modifications and optional variations of the concepts described in the present invention, and it is considered that such variations and modifications are within the scope of this invention as defined by the description and the appended claims. Furthermore, in case the features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also described in terms of any single element or subset of elements of the invention. Markush group or another group. The other embodiments are within the scope of the following claims.

TABLE I RNA synthesis cycle 0.2 μmol Reagents Equivalent Quantities (_L) Waiting time (sec) Phosphoramidites 15 31 465 SET 38.7 31 465 Acetic anhydride 655 124 5 N-methyl-imidazole 1245 124 5 TCA 700 732 10 Iodine 20.6 244 15 The waiting time does not include the contact time during the supply.

TABLE II Linear antisense nucleic acid molecules that select C-raf as target with no sequence matings Caption: Lowercase letters - 2'-0-metyribonucleotides Capital letters - deoxy-ribonucleotides s - phosphorothioate link TABLE III HPH molecules that target c-raf RNA with no matings I KNOW THAT. ID. HPH sequence No. RPI Length No. 5'-complementary RNA arm DNA center Complementary 3 'RNA arm 1 gscsgsagc gugguca GsCsGsTsGsCsAsAsGs cauugau gcuscsgsc 12020 35 2 gscsgsagc ggguacu JsCsGsGsAsCsAsGsGs uacuuag gcuscsgsc 12023 35 3 csgsasgc gugguca GsCsGsTsGsCsAsAsGs cauugau gcsuscsg 12021 I -1 33 O OO 4 csgsasgc ggguacu JsCsGsGsAsCsAsGsGs uacuuag gcsuscsg 12024 33 5 csasgsc gugguca GsCsGsTsGsCsAsAsGs cauugau gscsusg 12022 31 6 csasgsc ggguacu IsCsGsGsAsCsAsGsGs uacuuag gscsusg 12025 31 Caption: Lowercase letters 2'-0-methyl ribonucleotides Uppercase letters deoxy-ribonucleotides phosphorothioate link underlined no matings TABLE IV HPH molecules that target IMPDH and c-Raf RNA O lO Caption: Lowercase letters - 2'-0-methyl ribonucleotides Capital letters - deoxyribonucleotides s - phosphorothioate B - 3'-3 'or 5'-5'-deoxinucleotide inverted abyssal linkage Underlined - tempered region Negritas - non-pairing SCR target - Region self-complementary

Claims (12)

1. NOVELTY OF THE INVENTION CLAIMS 1. - A method for modulating the function of a target sequence in a cell comprising the step of contacting said cell with a hairpin hybridizing nucleic acid (HPH) molecule under appropriate conditions to modulate the expression of said target sequence, characterized in that said hairpin hybridizing nucleic acid molecule consists of the formula I: in which, each P, Y, N and M independently represent a nucleotide that could be the same or different; • indicates the formation of hydrogen bonds between two adjacent nucleotides; N 'is a complementary nucleotide for N; or is an integer greater than or equal to 3; w is an integer greater than or equal to 4; k and t are independently zero or an integer greater than or equal to 3; wherein when t or k are independently 3 or more, at least one of said P is not a nucleotide containing 2'-H; each of (P) t and (P) k includes internucleotide links that selected from the group consisting of phosphodiester, phosphorothioate, phosphorodithioate, 5'-thiophosphate, and methylphosphonate; (M) w is an oligonucleotide that includes one or more internucleotide bonds that are selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, methylphosphonate, and phosphorodithioate bonds, in which at least one of each said (P) t (P) k, and (M) w is an oligonucleotide of sufficient length to interact stably with the target sequence; r and f are independently an integer greater than or equal to zero; each B and B 'independently represent a blocking structure, which independently could be present or absent; and represents a chemical bond.
2. 2. The method according to claim 1, further characterized in that k in said hairpin hybridization nucleic acid molecule is less than 100.
3. 3. The method according to claim 2, further characterized in that k in said molecule of The hairpin hybridizer nucleic acid is selected from the group consisting of 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, and 20.
4. 4. The method according to claim 1, further characterized in that t in said hairpin hybridizing nucleic acid molecule is less than 100.
5. 5. The method according to claim 4, further characterized in that t in said hairpin hybridizing nucleic acid molecule is selected from the group consisting of 4.5. , 6, 7, 8, 9, 10, 11, 12, 15, and 20.
6. 6. The method according to claim 1, further characterized in that k and t in said hairpin hybridizing nucleic acid molecule are of the same length.
7. 7. The method according to claim 1, further characterized in that k and t in said fork hybridizing nucleic acid molecule are of different length.
8. 8. The method according to claim 1, further characterized in that w in said hairpin hybridizer nucleic acid molecule is less than 100.
9. 9. The method according to claim 8, further characterized in that w in said molecule of The hairpin hybridizing nucleic acid is selected from the group consisting of 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, and 20.
10. 10. The method according to claim 1, further characterized in that t, k, and w in said hairpin hybridizing nucleic acid molecule are of the same length.
11. 11. The nucleic acid molecule method according to claim 1, further characterized in that t, k, and w in said hairpin hybridizing nucleic acid molecule are of different length.
12. 12. The method according to claim 1, further characterized in that the target sequence is selected from the group consisting of RNA, DNA and mixed RNA / DNA polymers. 13. - The method according to claim 1, further characterized in that r and f in said hairpin hybridizing nucleic acid molecule are independently selected from the group consisting of, 2, 3, 4, 5, 10, and 15. 14. The method according to claim 1, further characterized in that the chemical bond in said hairpin hybridizing nucleic acid molecule is selected from the group consisting of Phosphate ester, amide bond, phosphorothioate, 5'-thiophosphate, methylphosphonate and phosphorodithioate. 15. A method for modulating the function of an objective sequence in a cell comprising the step of contacting said cell with a nucleic acid molecule. hairpin hybridizer (HPH) under appropriate conditions to modulate the function of said target sequence, characterized in that said hairpin hybridizing nucleic acid molecule consists of the formula II: in which, each P, N and M independently represent a nucleotide that could be the same or different; • indicates the formation of hydrogen bonds between two adjacent nucleotides; N 'is a complementary nucleotide for N; or is an integer greater than or equal to 3; w is an integer greater than or equal to 4; k, t, ki and ti are independently zero or an integer greater than or equal to 3; wherein when t, k, ti, and ki are independently 3 or more, at least one of said P is not a nucleotide containing 2'-H; each of (P) t, (P) k (P) t? and (P) ki independently include internucleotide linkages that are selected from the group consisting of phosphodiester, phosphorothioate, phosphorodithioate, 5'-thiophosphate, and methylphosphonate; (M) w is an oligonucleotide sequence that includes one or more internucleotide linkages that are selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, methylphosphonate and phosphorodithioate bonds, in which at least one of each said (P ) t, (P) k (P) n > (P) ki. and (M) is an oligonucleotide of sufficient length to interact stably independently with the target sequence; and represents a chemical bond. 16. The method according to claim 15, further characterized in that each of k, t, k1, t1 and w in said fork hybridizing molecule is less than 100. 17. The method according to claim 16, characterized in addition because each of k, t, k1, t1 and w in said hairpin hybridizing nucleic acid molecule are independently selected from the group consisting of 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, and 20. 18. The method according to claim 15, further characterized in that k and t in said hairpin hybridizing nucleic acid molecule are of the same length. 19. The method according to claim 15, further characterized in that k and t in said hairpin hybridizing nucleic acid molecule are of different length. 20. The method according to claim 15, further characterized in that t, k and w in said hairpin hybridizing nucleic acid molecule are of the same length. 21. The method according to claim 15, further characterized in that t, k and w in said hairpin hybridizing nucleic acid molecule are of different length. 22. The method according to claim 15, further characterized in that t1, k1 and w in said hairpin hybridizing nucleic acid molecule are of the same length. 23. The method according to claim 15, further characterized in that t1, k1 and w in said hairpin hybridizing nucleic acid molecule are of different length. 24. The method according to claim 15, further characterized in that the target sequence is selected from the group consisting of RNA, DNA and mixed RNA / DNA polymers. 25. The method according to claim 15, further characterized in that the chemical bond in said hairpin hybridizing nucleic acid molecule is selected from the group consisting of phosphate ester bond, amide bond, phosphorothioate, 5'-thiophosphate, methylphosphonate and phosphorodithioate, 26.- A method for modulating the function of an objective sequence in a cell comprising the step of contacting said cell with a molecule of nucleic acid hairpin hybridizer (HPH) under appropriate conditions to modulate the function of said target sequence, characterized in that said hairpin hybridizing nucleic acid molecule consists of the formula III: B B 'in which, each P, N and M independently represent a nucleotide that could be the same or different; • indicates the formation of hydrogen bonds between two adjacent nucleotides; N 'is a complementary nucleotide for N; or is an integer greater than or equal to 3; w is an integer greater than or equal to 4; k and t are independently zero or an integer greater than or equal to 3; wherein when t and k are independently 3 or more, at least one of said P is not a nucleotide containing 2'-H; each of (P) t and (P) k include internucleotide linkages that are selected from the group consisting of phosphodiester, phosphorothioate, phosphorodithioate, 5'-thiophosphate, and methylphosphonate; (M) w is an oligonucleotide sequence that includes one or more internucleotide linkages that are selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, methylphosphonate and phosphorodithioate linkages; D and E are oligonucleotides which independently have length greater than or equal to 4; wherein at least one or more of said (P) t, (P) k (P) t- ?, (M) w, D and E is independently an oligonucleotide of sufficient length to independently interact stably with the target nucleic acid sequence; B and B 'independently represent a blocking structure, which independently could be present or absent; and represents a chemical bond. 27. The method according to claim 26, further characterized in that each k, t, and w in said hairpin hybridizing nucleic acid molecule is independently less than 100. 28.- The method according to claim 27, further characterized because each k, t, and w in said hairpin hybridizing nucleic acid molecule is selected from the group consisting of 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, and 20. 29.- The method according to claim 26, further characterized in that k and t in said hairpin hybridizing nucleic acid molecule are of the same length. 30. The method according to claim 26, further characterized in that k and t in said hairpin hybridizing nucleic acid molecule are of different length. 31. The method according to claim 26, further characterized in that each t, k and w in said hairpin hybridizing nucleic acid molecule are of the same length. 32. The method according to claim 26, further characterized in that each t, k and w in said hairpin hybridizing nucleic acid molecule are of the same length. 33. The method according to claim 26, further characterized in that the target nucleic acid sequence is selected from the group consisting of RNA, DNA and mixed RNA / DNA polymers. 34. The method according to claim 26, further characterized in that each oligonucleotide D and E is independently less than 100 nucleotides in length. The method according to claim 26, further characterized in that each oligonucleotide D and E is independently selected from the group consisting of 6, 7, 8, 9, 10, 11, 15, 20, and 30 nucleotides. 36. The method according to claim 26, further characterized in that each oligonucleotide D and E in said fork hybridizing nucleic acid molecule are of the same length. 37. The method according to claim 26, further characterized in that said oligonucleotides, said oligonucleotide D and said oligonucleotide E in said hairpin hybridizing nucleic acid molecule are of different length. 38. The method according to claim 26, further characterized in that said chemical bond is selected from the group consisting of phosphate ester linkage, amide bond, phosphorothioate, and phosphorodithioate, 39.- A method for modulating the function of a target sequence in a cell comprising the step of contacting said cell with a hairpin hybridizing nucleic acid (HPH) molecule under appropriate conditions to modulate the function of said target sequence, characterized in that said hairpin hybridizing nucleic acid molecule consists of the Formula V: in which, each P, N, F, V, Z and M independently represent a nucleotide that could be the same or different; • indicates the formation of hydrogen bonds between two adjacent nucleotides; N 'is a complementary nucleotide for N; F 'is a complementary nucleotide for F; or is an integer greater than or equal to 3; w is an integer greater than or equal to 4; d is an integer greater than or equal to 3; h is a whole number greater than or equal to 2; c is an integer greater than or equal to 0; k and t are independently zero or an integer greater than or equal to 3; wherein when t and k are independently 3 or more, at least one of said P is not a nucleotide containing 2'-H; each (P) t, (P) k, (V) d, and (Z) c include n-nucleotide linkages that are selected from the group consisting of phosphodiester, phosphorothioate, phosphorodithioate, 5'-thiophosphate, and methylphosphonate; (M) w is an oligonucleotide that includes one or more internucleotide linkages that are selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, methylphosphonate and phosphorodithioate linkages, wherein at least one or more of each said (P) t, (P) k and (M) w is an oligonucleotide of sufficient length to stably interact independently with an objective sequence; each B and B 'independently represent a blocking structure, which independently could be present or absent; and represents a chemical bond. 40.- A method for modulating the function of a target sequence in a cell comprising the step of contacting said cell with a hairpin hybridizing nucleic acid (HPH) molecule under appropriate conditions to modulate the function of said target sequence, characterized in that said hairpin hybridizing nucleic acid molecule consists of the formula VI: in which, each P, N, F, Z and M independently represent a nucleotide that could be the same or different; • indicates the formation of hydrogen bonds between two adjacent nucleotides; N 'is a complementary nucleotide for N; F 'is a complementary nucleotide for F; or is an integer greater than or equal to 3; k and t are independently zero or an integer greater than or equal to 3; k1 and t1 are independently zero or an integer greater than or equal to 3; w is an integer greater than or equal to 4; d is an integer greater than or equal to 3; h is an integer greater than or equal to 2; c is an integer greater than or equal to 0; wherein when t, k, t1 or k1 are independently 3 or more, at least one of said P is not a nucleotide containing 2'-H; each (P) t, (P) k, (P) t ?, (P) M, and (Z) c include internucleotide linkages that are selected from the group consisting of phosphodiester, phosphorothioate, phosphorodithioate, 5'-thiophosphate, and methylphosphonate; (M) w is an oligonucleotide that includes one or more internucleotide bonds that are selected from the group consisting of links phosphodiester, phosphorothioate, 5'-thiophosphate, methylphosphonate and phosphorodithioate, in which at least one or more of each of said (P) t, (P) k, (P) t ?. (P) M and (M) w is an oligonucleotide of sufficient length to interact stably independently with an objective sequence; each B and B 'independently represent a blocking structure, which independently could be present or absent; and represents a chemical bond. 41. A method for modulating the function of an objective sequence in a cell comprising the step of contacting said cell with a hairpin hybridizing nucleic acid (HPH) molecule under appropriate conditions to modulate the function of said target sequence, characterized in that said hairpin hybridizing nucleic acid molecule consists of the formula VII: in which, each P, N, F, V, Z and M independently represent a nucleotide that could be the same or different; • indicates the formation of hydrogen bonds between two adjacent nucleotides; N 'is a nucleotide complementary to N; F 'is a complementary nucleotide for F; or is an integer greater than or equal to 3; w is an integer greater than or equal to 4; d is an integer greater than or equal to 3; h is an integer greater than or equal to 2; c is an integer greater than or equal to 0; k and t are independently zero or an integer greater than or equal to 3; wherein when t and k are independently 3 or more, at least one of said P is not a nucleotide containing 2'-H; each (P) t, (P) k, (V) d, and (Z) c include internucleotide linkages that are selected from the group consisting of phosphodiester, phosphorothioate, phosphorodithioate, 5'-thiophosphate, and methylphosphonate; (M) w is an oligonucleotide that includes one or more internucleotide bonds that are selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, methylphosphonate, and phosphorodithioate bonds, wherein at least one or more of each said (P) t, (P) k and (M) w is an oligonucleotide of sufficient length to interact stably independently with an objective sequence; each B and B 'independently represent a blocking structure, which independently could be present or absent; and represents a chemical bond. 42. The method according to any of claims 39-41, further characterized in that each k, t, and w in said hairpin hybridizing nucleic acid molecule is independently less than 100. 43.- The method of compliance with claim 42, further characterized in that each k, t, and w in said acid molecule nucleic hairpin hybridizer is independently selected from the group consisting of 4, 5, 6, 7, 8, 9, 10, 11, 12, 15 and 20. 44.- The method according to any of claims 39- 41, further characterized in that d in said hairpin hybridizing nucleic acid molecule is independently selected from the group consisting of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16 and 18. 45. The method according to any of claims 39-41, further characterized in that h in said hairpin hybridizing nucleic acid molecule is independently selected from the group consisting of 2, 3, 4, 5, 6, 7, 8 and 9. 46. The method according to any of claims 39-41, further characterized in that c in said hairpin hybridizing nucleic acid molecule is independently selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16 and 18. 47. The method according to any of claims 39-41, further characterized in that or in said hairpin hybridizing nucleic acid molecule is selects independently from the group consisting of 4, 5, 6, 7, 8 and 9. 48. The method according to any of claims 39-41, further characterized in that k and t in said hairpin hybridizing nucleic acid molecule are of the same length. 49.- The method of compliance with any of the claims 39-41, further characterized in that k and t in said hairpin hybridizing nucleic acid molecule are of different length. 50. The method according to any of claims 39-41, further characterized in that each t, k and w in said fork hybrid nucleic acid molecule are of different length. 51. The method according to any of claims 39-41, further characterized in that each t, k and w in said hairpin hybridizing nucleic acid molecule are of the same length. 52. The method according to any of claims 39-41, further characterized in that the target sequence is selected from the group consisting of RNA, DNA and mixed RNA / DNA polymers. 53. The method according to any of claims 39-41, further characterized in that the chemical bond is selected from the group consisting of phosphate ester bond, amide bond, phosphorothioate, 5'-thiophosphate, methylphosphonate and phosphorodithioate, 54 The method according to claim 40, further characterized in that each k1 and t1 in said hairpin hybridizing nucleic acid molecule is independently less than 100. The method according to claim 40, further characterized in that each k1 and t1 in said hairpin hybridizing nucleic acid molecule is independently selected from the group consisting of 4, 5, 6, 7, 8, 9, 10, 11, 12, 15 and 20. 56. - The method according to claim 40, further characterized in that each t1, k1 and w in said hairpin hybridizing nucleic acid molecule is of different length. 57. The method according to claim 40, further characterized in that each t1, k1 and w in said fork hybridizing nucleic acid molecule is of equal length. 58. The method according to any of claims 39-41, further characterized in that said portion F of (F »F ') h in the HPH nucleic acid molecule is complementary to a portion of said target sequence. 59. The method according to any of claims 39-41, further characterized in that said portion F 'of (F »F') h in the HPH nucleic acid molecule is complementary to a portion of said target sequence. The method according to any of claims 39-41, further characterized in that said portion F and said portion F 'of (F «F') h in the nucleic acid molecule HPH is complementary to said sequence independently objective. 61.- The method according to any of claims 1, 15, 26, or 39-41, further characterized in that said portion N of (N * N ') 0 in the HPH nucleic acid molecule is complementary to a portion of said objective sequence. 62.- The method of compliance with any of the claims 1, 15, 26, or 39-41, further characterized in that said portion N 'of (N »N') 0 in the HPH nucleic acid molecule is complementary to said target sequence. 63. The method according to any of claims 1, 15, 26 or 39-41, further characterized in that said portion N and said portion N 'of (N * N') 0 in the nucleic acid molecule HPH is complementary independently of said objective sequence. 64.- The method according to any of claims 39-41, further characterized in that said (Z) c in the HPH nucleic acid molecule is complementary to an objective sequence. The method according to any of claims 1, 15, 26 or 39-41, further characterized by each of said (P) k, (P) t, (N »N ') 0, and (M) ) w, in the nucleic acid molecule HPH independently comprises a nucleotide modification which is selected from the group consisting of 2'-0-methyl, 2'-0-allyl, 21-0-methylthiomethyl, L-nucleotides, 2'-C-allyl, 1-5-anhydrohexitol; 2,6-diaminopurine; 2'-fluoro; 2'-deoxy-2'-amino; 2 '- (? / - alanyl) amino; 2 '- (N-phenylalanyl) amino; 21-deoxy-2 '- (N-beta-alanyl) amino; 2'-deoxy-2 '- (lysyl) amino; 2'-0-amino; 2'-deoxy-.2 '- (A / -histidyl) amino; 6-methyluridine; 5-methylcytidine; 2 '- (? / - b-carboxamidin-beta-aIanyl) amino-2'-deoxynucleotide; and xylofuranosyl. 66.- The method according to claim 1, further characterized in that each of said (Y) r and (Y) f in the HPH nucleic acid molecule independently comprises a modification of nucleotide selected from the group consisting of 2'-0-methyl, 2'-0-allyl, 2'-0-methyltomethyl, L-nucleotides, 2'-C-allyl, 1-5-anhydrohexitol; 2,6-diaminopurine; 2'-fluoro; 2'-deoxy-2'-amino; 2 '- (? / - alanyl) amino; 2 '- (N-phenylalanyl) amino; 2'-deoxy-2 '- (N-beta-alanyl) amino; 2'-deoxy-2 '- (lysyl) amino; 21-O-amino; 2'-deoxy-2 '- (? / - histidyl) amino; 6-methyluridine; 5-methylcytidine; 2'-. { N-b-carboxamidin-beta-alanyl) amino-2'-deoxynucleotide; and xylofuranosyl. 67.- The method according to any of claims 15 or 40, further characterized by each of said (P) M and (P) t? in the nucleic acid molecule HPH independently comprises a nucleotide modification which is selected from the group consisting of 2'-O-methyl, 2'-0-allyl, 2'-0-methylthiomethyl, L-nucleotides, 2 ' -C-allyl, 1-5-anhydrohexitoi; 2,6-diaminopurine; 2'-fluoro; 2'-deoxy-2'-amino; 21 - (? / - alanyl) amino; 2 '- (N-phenylalanyl) amino; 2'-deoxy-2 '- (N-beta-alanyl) amino; 21-deoxy-2 '- (lysyl) amino; 2'-0-amino; 2'-deoxy-2 '- (N-histidyl) amino; 6-methyluridine; 5-methyclitidine; 2 '- (/ V-b-carboxamidin-beta-alanyl) amino-2'-deoxynucleotide; and xylofuranosyl. 68.- The method according to claim 26, further characterized in that each of said D and E in the HPH nucleic acid molecule independently comprises a nucleotide modification that is selected from the group consisting of 2'-0- methyl, 2'-0-allyl, 2'-0-methylthiomethyl, L-nucleotides, 2'-C-allyl, 1-5-anhydrohexitol; 2,6-diaminopurine; 2'-fluoro; 2'-deoxy-2'-amino; 2 '- (? / - alanyl) amino; 2 '- (N-phenylalanyl) amino; 2'-deoxy-2 '- (N-beta-alanyl) amino; 2'-deoxy-2 '- (lysyl) amino; twenty-one- O-amino; 2'-deoxy-2 '- (/ V-histidyl) amino; 6-methyluridine; 5-methylcytidine; 2 '- (/ V-b-carboxamidin-beta-alanyl) amino-2'-deoxynucleotide; and xylofuranosyl. 69. The method according to any of claims 39-41, further characterized in that each of said (Z) cy (F "F ') h in the HPH nucleic acid molecule independently comprises a nucleotide modification that is selected from the group consisting of 2'-0-methyl, 2'-0-allyl, 2'-0-methylthiomethyl, L-nucleotides, 2'-C-allyl, 1-5-anhydrohexitol; 2,6-diaminopurine; 2'-fluoro; 2'-deoxy-2'-amino; 21 - (? - alanyl) amino; 2 '- (N-phenylalanyl) amino; 2'-deoxy-2 '- (N-beta-alanyl) amino; 21-deoxy-2 '- (lysyl) amino; 2'-0-amino; 2'-deoxy-2 '- (? / - histidyl) amino; 6-methyluridine; 5-methylcytidine; 2 '- (? / - b-carboxamidin-beta-alanyl) amino-2'-deoxynucleotide; and xylofuranosyl. The method according to any of claims 39 or 41, further characterized in that each of said (V) d in the HPH nucleic acid molecule independently comprises a nucleotide modification that is selected from the group consisting of 2'-0-methyl, 2'-0-allyl, 2'-0-methylthiomethyl, L-nucleotides, 2'-C-allyl, 1-5-anhydrohexitol; 2,6-diaminopurine; 2'-fluoro; 2'-deoxy-2'-amino; 21 - (? / - alanyl) amino; 2 '- (N-phenylalanyl) amino; 2'-deoxy-2 '- (N-beta-alanyl) amino; 21-deoxy-2 '- (lysyl) amino; 2'-0-amino; 2'-deoxy-2 '- (A / -histidyl) amino; 6-methyluridine; 5-methylcytidine; 2 '- (N-b-carboxamidin-beta-alaniI) amino-2'-deoxynucleotide; and xylofuranosyl. 71.- The method of compliance with any of the claims 1, 26 or 39-41, further characterized in that said B ', when present is selected from the group consisting of inverted abbasic residue, 4' nucleotide, 5'-methylene, nucleotide I- (beta-D-erythrofuranosyl), nucleotide 4'-thio, carbocyclic nucleotide; 1, 5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; nucleotide of modified base; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; 3 ', 4'-non-cyclic dry nucleotide; non-cyclic 3,4-dihydroxybutyl nucleotide; 3,4-dihydroxypentyl nucleotide non-cyclic; 3'-3'-inverted nucleotide portion; 3'-3'-inverted abasic portion; 3'-2'-inverted nucleotide portion; 3'-2'-inverted abasic portion; 1,4-butanediol phosphate; 3'-phosphoramidate; hexyl phosphate; aminohexylphosphate; 3'-phosphate; 3'-phosphorothioate; phosphorodithioate; and methylphosphonate moiety. The method according to any of claims 1, 26 or 39-41, further characterized in that said nucleic acid molecule comprises an inverted abasic portion with 3'-3 'linkage at said 3' end. 73. The method according to any of claims 1, 26 or 39-41, further characterized in that said B, when present is selected from the group consisting of nucleotide 4 ', 5'-methylene, nucleotide I- (beta -D-eritrofuranosium), nucleotide 4'-thio, carbocyclic nucleotide; 5'-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1, 2-aminododecyl phosphate; hydroxypropyl phosphate; 1, 5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; nucleotide of modified base; phosphorodithioate; Ireo-pentofuranosyl nucleotide; 3 ', 4'-non-cyclic dry nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide; 5'-5'-inverted nucleotide portion; 5'-5'-inverted abasic portion; 5'-phosphoramide; 5'-phosphorothioate; 1,4-butanediol phosphate; 5'-amino; 5'-phosphoramidate bridging or non-bridging; phosphorothioate and / or phosphorodithioate, portions of methylphosphonate and 5'-mercapto bridging or non-bridging. 74. The method according to any of claims 1, 15, 26 or 39-41, further characterized in that said cell is a mammalian cell. The method according to any of claims 1, 15, 26 or 39-41, further characterized in that said cell is a plant cell. The method according to any of claims 1, 15, 26 or 39-41, further characterized in that said cell is a bacterial cell. 77. The method according to any of claims 1, 15, 26 or 39-41, further characterized in that said cell is a microbial cell. 78. The method according to any of claims 1, 15, 26 or 39-41, further characterized in that said cell is a fungal cell. 79.- The mammalian cell in accordance with the claim 74, further characterized in that said mammalian cell is a human cell. 80. The method according to any of claims 1, 15, 26 or 39-41, further characterized in that said HPH nucleic acid molecule is chemically synthesized. 81. The method according to any of claims 1, 15, 26 or 39-41, further characterized in that said HPH is a pharmaceutical composition. 82. The method according to any of claims 1, 15, 26 or 39-41, further characterized in that said modulation of the function is the modulation of the phenotype of the cell. 83.- A method for modulating the function of an objective sequence in a cell comprising the step of contacting said cell with a hairpin hybridizing nucleic acid (HPH) molecule under appropriate conditions to modulate the function of said target sequence, characterized in that said hairpin hybridizing nucleic acid molecule consists of the formula IX: wherein, each F, D, O, K, W, and T independently represents an oligonucleotide whose nucleotide sequence could be the same or different; could be of the same or different length and could include, individually or in combination, internucleotide linkages that are selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, methylphosphonate; F and D independently or in combination form an RNAse H activation domain, wherein F and D are of greater than or equal to 4 nucleotides; • indicates the formation of hydrogen bonds between two adjacent nucleotides within the HPH nucleic acid molecule; D comprises the nucleotide sequence that is complementary to the nucleotide sequence of O; K comprises the nucleotide sequence that is complementary to the nucleotide sequence of T; D and O form base pairs greater than or equal to two with each other which are contiguous or non-contiguous; K and T form base pairs greater than or equal to two with each other which are contiguous or non-contiguous; the paired base regions K «T and O» D within the HPH nucleic acid molecule could be contiguous or non-contiguous with one another; K, T, O and W are of greater length than or equal to 3 nucleotides; F, D, K, T, W and O together are of sufficient length to interact stably with the target sequence; each B and B 'independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond. 84.- A method to modulate the function of a sequence target in a cell comprising the step of contacting said cell with a hairpin hybridizing nucleic acid (HPH) molecule under appropriate conditions to modulate the function of said target sequence, characterized in that said hairpin hybridizing nucleic acid molecule consists of the formula X: B B ' in which, each F, D, O, K, W, and T independently represents an oligonucleotide whose nucleotide sequence could be the same or different; could be of the same or different length and could include, individually or in combination, internucleotide linkages that are selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, methylphosphonate; F and D independently form an RNAse H activation domain, where F and D are greater than or equal to 4 nucleotides; • indicates the formation of hydrogen bonds between two adjacent nucleotides within the HPH nucleic acid molecule; D comprises the nucleotide sequence that is complementary to the nucleotide sequence of O; K comprises the nucleotide sequence that is complementary to the nucleotide sequence of T; D and O form base pairs greater than or equal to two with each other that are contiguous or not contiguous; K and T form base pairs greater than or equal to two with each other which are contiguous or non-contiguous; the regions of paired bases K «T and O» D could be contiguous or not contiguous with each other; K, T, O and W are of greater length than or equal to 3 nucleotides; F, D, K, T, W and O together are of sufficient length to interact stably with the target sequence; each B and B 'independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond. 85.- A method for modulating the function of a target sequence in a cell comprising the step of contacting said cell with a hairpin hybridizing nucleic acid (HPH) molecule under appropriate conditions to modulate the function of said target sequence, characterized in that said hairpin hybridizing nucleic acid molecule consists of the formula XI: wherein, each F, D, O, K, W, and T independently represents an oligonucleotide whose nucleotide sequence could be the same or different; could be of the same length or different length and could include, individually or in combination, internucleotide links that are selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, methylphosphonate; F and D independently or in combination form an RNAse H activation domain, wherein F and D are of greater than or equal to 4 nucleotides; • indicates the formation of hydrogen bonds between two adjacent nucleotides within the HPH nucleic acid molecule; D comprises the nucleotide sequence that is complementary to the nucleotide sequence of O; K comprises the nucleotide sequence that is complementary to the nucleotide sequence of T; D and O form base pairs greater than or equal to two with each other which are contiguous or non-contiguous; K and T form base pairs greater than or equal to two with each other which are contiguous or non-contiguous; the base-paired regions K * T and O »D within the HPH nucleic acid molecule could be contiguous or non-contiguous with one another; K, T, O and W are of greater length than or equal to 3 nucleotides; F, D, K, T, W and O together are of sufficient length to interact stably with the target sequence; each B and B 'independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond. 86.- A method for modulating the function of an objective sequence in a cell comprising the step of contacting said cell with a hairpin hybridizing nucleic acid (HPH) molecule under appropriate conditions to modulate the function of said target sequence, characterized in that said hybridizing nucleic acid molecule of fork consists of the formula XII: B B 'I I D-0 w in which, each D, O and W independently represents an oligonucleotide whose nucleotide sequence could be the same or different; could be of the same or different length and could include, individually or in combination, internucleotide linkages that are selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, methylphosphonate; D independently forms an RNAse H activation domain of greater than or equal to 4 nucleotides; • indicates the formation of hydrogen bonds between two adjacent nucleotides within the HPH nucleic acid molecule; D comprises the sequence of nucieotides that is complementary to the nucleotide sequence of O; D and O form pairs of bases greater or equal to two with each other that are contiguous or non-contiguous; O and W are of greater length than or equal to 3 nucleotides; D, W and O together are of sufficient length to interact stably with the target sequence; each B and B 'independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond. 87. A method for modulating the function of a target sequence in a cell comprising the step of contacting said cell with a hairpin hybridizing nucleic acid (HPH) molecule under appropriate conditions to modulate the function of said target sequence, characterized in that said hairpin hybridizing nucleic acid molecule consists of the formula XIII: B B 'I I O- D W in which, each D, O and W independently represents an oligonucleotide whose nucleotide sequence could be the same or different; could be of the same or different length and could include, individually or in combination, internucleotide linkages that are selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, methylphosphonate; D independently forms an RNAse H activation domain of greater than or equal to 4 nucleotides; • indicates the formation of hydrogen bonds between two adjacent nucleotides within the HPH nucleic acid molecule; D comprises the nucleotide sequence that is complementary to the nucleotide sequence of O; D and O form base pairs greater than or equal to two with each other which are contiguous or non-contiguous; O and W are of greater length than or equal to 3 nucleotides; D, W and O together are of sufficient length to interact stably with the target sequence; each B and B 'independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond. 88.- A method for modulating the function of an objective sequence in a cell comprising the step of contacting said cell with a hairpin hybridizing nucleic acid (HPH) molecule under appropriate conditions to modulate the function of said target sequence, characterized in that said hairpin hybridizing nucleic acid molecule consists of the formula XIV: B B 'i A I O - D I I K - T \ / w wherein, each A, D, O, K, W, and T independently represents an oligonucleotide whose nucleotide sequence could be the same or different; could be of the same or different length and could include, individually or in combination, internucleotide linkages that are selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, methylphosphonate; D independently forms a domain of RNase H activation of greater than or equal to 4 nucleotides; • indicates the formation of hydrogen bonds between two adjacent nucleotides within the HPH nucleic acid molecule; D comprises the nucleotide sequence that is complementary to the nucleotide sequence of O; K comprises the nucleotide sequence that is complementary to the nucleotide sequence of T; D and O form base pairs greater than or equal to two with each other which are contiguous or non-contiguous; K and T form base pairs greater than or equal to two with each other which are contiguous or non-contiguous; the regions of paired bases K »T and 0» D could be contiguous or non-contiguous with each other; A, K, T, O and W are of greater length than or equal to 3 nucleotides; A, D, K, T, W and O together are of sufficient length to interact stably with the target sequence; each B and B 'independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond. 89.- A method for modulating the function of an objective sequence in a cell comprising the step of contacting said cell with a hairpin hybridizing nucleic acid (HPH) molecule under appropriate conditions to modulate the function of said target sequence, characterized in that said hairpin hybridizing nucleic acid molecule consists of the formula XV: B B 'I A o. KA wherein, each A, D, O, K, W, and T independently represents an oligonucleotide whose nucleotide sequence could be the same or different; could be of the same or different length and could include, individually or in combination, internucleotide linkages that are selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, methylphosphonate; D independently forms an RNAse H activation domain of greater than or equal to 4 nucleotides; • indicates the formation of hydrogen bonds between two adjacent nucleotides within the HPH nucleic acid molecule; D comprises the nucleotide sequence that is complementary to the nucleotide sequence of O; K comprises the nucleotide sequence that is complementary to the nucleotide sequence of T; D and O form base pairs greater than or equal to two with each other which are contiguous or non-contiguous; K and T form base pairs greater than or equal to two with each other which are contiguous or non-contiguous; the regions of paired bases K T and O »D could be contiguous or non-contiguous with each other; A, K, T, O and W are of greater length than or equal to 3 nucleotides; A, D, K, T, W and O together are of sufficient length to interact stably with the target sequence; each B and B 'independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond. 90.- A method for modulating the function of an objective sequence in a cell comprising the step of contacting said cell with a hairpin hybridizing nucleic acid (HPH) molecule under conditions appropriate for modulating the function of said target sequence, characterized in that said hairpin hybridizing nucleic acid molecule consists of the formula XVI: B B 'i A I D - O I I K - T \ / w wherein, each A, D, O, K, W, and T independently represents an oligonucleotide whose nucleotide sequence could be the same or different; could be of the same or different length and could include, individually or in combination, internucleotide linkages that are selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, methylphosphonate; D independently forms an RNAse H activation domain of greater than or equal to 4 nucleotides; • indicates the formation of hydrogen bonds between two adjacent nucleotides within the HPH nucleic acid molecule; D comprises the nucleotide sequence that is complementary to the nucleotide sequence of O; K comprises the nucleotide sequence that is complementary to the nucleotide sequence of T; D and O form base pairs greater than or equal to two with each other which are contiguous or non-contiguous; K and T form base pairs greater than or equal to two with each other which are contiguous or non-contiguous; the regions of paired bases KVT and O * D could be contiguous or non-contiguous with each other; A, K, T, O and W are of greater length than or equal to 3 nucleotides; A, D, K, T, W and O together are of sufficient length to interact stably with the target sequence; each B and B 'independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond. 91.- A method for modulating the function of an objective sequence in a cell comprising the step of contacting said cell with a hairpin hybridizing nucleic acid (HPH) molecule under appropriate conditions to modulate the function of said target sequence, characterized in that said hairpin hybridizing nucleic acid molecule consists of formula XVII: wherein, each A, D, O, K, W, and T independently represents an oligonucleotide whose nucleotide sequence could be the same or different; it could be of the same length or of different length and could include, individually or in combination, internucleotide bonds which are selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, and methylphosphonate; D independently forms an RNAse H activation domain of greater than or equal to 4 nucleotides; • indicates the formation of hydrogen bonds between two adjacent nucleotides within the HPH nucleic acid molecule; D comprises the nucleotide sequence that is complementary to the nucleotide sequence of O; K comprises the nucleotide sequence that is complementary to the nucleotide sequence of T; D and O form base pairs greater than or equal to two with each other which are contiguous or non-contiguous; K and T form greater or equal pairs of bases to two with each other that are contiguous or non-contiguous; the regions of paired bases K «T and O» D could be contiguous or not contiguous with each other; A, K, T, O and W are of greater length than or equal to 3 nucleotides; A, D, K, T, W and O together are of sufficient length to interact stably with the target sequence; each B and B 'independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond. 92.- A method for modulating the function of an objective sequence in a cell comprising the step of contacting said cell with a hairpin hybridizing nucleic acid (HPH) molecule under appropriate conditions to modulate the function of said target sequence, characterized in that said hairpin hybridizing nucleic acid molecule consists of the formula XVIII: B B 'I A 0- D W wherein, each A, D, O, and W independently represents an oligonucleotide whose nucleotide sequence could be the same or different; could be of the same or different length and could include, individually or in combination, internucleotide linkages that are selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, and methylphosphonate; D independently forms a domain of RNase H activation of greater than or equal to 4 nucleotides; • indicates the formation of hydrogen bonds between two adjacent nucleotides within the HPH nucleic acid molecule; D comprises the nucleotide sequence that is complementary to the nucleotide sequence of O; D and O form base pairs greater than or equal to two with each other which are contiguous or non-contiguous; A, O and W are of greater length than or equal to 3 nucleotides; A, D, W and O together are of sufficient length to interact stably with the target sequence; each B and B 'independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond. 93.- A method to modulate the function of a sequence target in a cell comprising the step of contacting said cell with a hairpin hybridizing nucleic acid (HPH) molecule under appropriate conditions to modulate the function of said target sequence, characterized in that said hairpin hybridizing nucleic acid molecule consists of the formula XIX: B B 'I A O- D W wherein, each A, D, O, and W independently represents an oligonucleotide whose nucleotide sequence could be the same or different; could be of the same or different length and could include, individually or in combination, internucleotide linkages that are selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, and methylphosphonate; D independently forms an RNAse H activation domain of greater than or equal to 4 nucleotides; • indicates the formation of hydrogen bonds between two adjacent nucleotides within the HPH nucleic acid molecule; D comprises the nucleotide sequence that is complementary to the nucleotide sequence of O; D and O form base pairs greater than or equal to two with each other which are contiguous or non-contiguous; A, O and W are of greater length than or equal to 3 nucleotides; A, D, W and O together are of sufficient length to interact stably with the target sequence; each B and B 'independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond. 94.- A method for modulating the function of an objective sequence in a cell comprising the step of contacting said cell with a hairpin hybridizing nucleic acid (HPH) molecule under appropriate conditions to modulate the function of said target sequence, characterized in that said hairpin hybridizing nucleic acid molecule consists of the formula XX: B B I F O- D W wherein, each F, D, O, and W independently represents an oligonucleotide whose nucleotide sequence could be the same or different; could be of the same or different length and could include, individually or in combination, internucleotide linkages that are selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, and methylphosphonate; F and D independently form an RNAse H activation domain of greater than or equal to 4 nucleotides; • indicates the formation of hydrogen bonds between two nucleotides adjacent to the HPH nucleic acid molecule; D comprises the nucleotide sequence that is complementary to the nucleotide sequence of O; D and O form base pairs greater than or equal to two with each other that are contiguous or non-contiguous; O and W are of greater length than or equal to 3 nucleotides; F, D, W and O together are of sufficient length to interact stably with the target sequence; each B and B 'independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond. 95.- A method for modulating the function of an objective sequence in a cell comprising the step of contacting said cell with a hairpin hybridizing nucleic acid (HPH) molecule under appropriate conditions to modulate the function of said target sequence, characterized in that said hairpin hybridizing nucleic acid molecule consists of the formula XXI: B B 'I F O- D W wherein, each F, D, O, and W independently represents an oligonucleotide whose nucleotide sequence could be the same or different; could be of the same length or different length and could include in individually or in combination, internucleotide linkages that are selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, and methylphosphonate; F and D independently or in combination form an RNAse H activation domain; • indicates the formation of hydrogen bonds between two adjacent nucleotides within the HPH nucleic acid molecule; D comprises the nucleotide sequence that is complementary to the nucleotide sequence of O; D and O form base pairs greater than or equal to two with each other which are contiguous or non-contiguous; O and W are of greater length than or equal to 3 nucleotides; F, D, W and O together are of sufficient length to interact stably with the target sequence; each B and B 'independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond. 96.- A method for modulating the function of an objective sequence in a cell comprising the step of contacting said cell with a hairpin hybridizing nucleic acid (HPH) molecule under appropriate conditions to modulate the function of said target sequence, characterized in that said hairpin hybridizing nucleic acid molecule consists of the formula XXII: wherein, each A, D, O, and W independently represents an oligonucleotide whose nucleotide sequence could be the same or different; could be of the same or different length and could include, individually or in combination, internucleotide linkages that are selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, and methylphosphonate; D independently forms an RNAse H activation domain of greater than or equal to 4 nucleotides; • indicates the formation of hydrogen bonds between two adjacent nucleotides within the HPH nucleic acid molecule; D comprises the nucleotide sequence that is complementary to the nucleotide sequence of O; D and O form base pairs greater than or equal to two with each other which are contiguous or non-contiguous; A, O and W are of greater length than or equal to 3 nucleotides; A, D, W and O together are of sufficient length to interact stably with the target sequence; each B and B 'independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond. 97.- A method to modulate the function of a sequence target in a cell comprising the step of contacting said cell with a hairpin hybridizing nucleic acid (HPH) molecule under appropriate conditions to modulate the function of said target sequence, characterized in that said hairpin hybridizing nucleic acid molecule consists of Formula XIII: B B 'I A D -0 W wherein, each A, D, O, and W independently represents an oligonucleotide whose nucleotide sequence could be the same or different; could be of the same or different length and could include, individually or in combination, intemucleotide bonds which are selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, and methylphosphonate; D independently forms an RNAse H activation domain of greater than or equal to 4 nucleotides; • indicates the formation of hydrogen bonds between two adjacent nucleotides within the HPH nucleic acid molecule; D comprises the nucleotide sequence that is complementary to the nucleotide sequence of O; D and O form base pairs greater than or equal to two with each other which are contiguous or non-contiguous; A, O and W are of greater length than or equal to 3 nucleotides; A, D, W and O together are of sufficient length to interact stably with the target sequence; each B and B 'independently represent an end biochemistry structure which independently could be present or absent; and represents a chemical bond. 98.- A method "for modulating the function of a target sequence in a cell comprising the step of contacting said cell with a hairpin hybridizing nucleic acid (HPH) molecule under appropriate conditions to modulate the function of said target sequence , characterized in that said hairpin hybridizing nucleic acid molecule consists of the formula XXIV: B B "I F D -O W wherein, each F, D, O, and W independently represents an oligonucleotide whose nucleotide sequence could be the same or different; could be of the same or different length and could include, individually or in combination, internucleotide linkages that are selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, and methylphosphonate; F and D independently or in combination form an RNAse H activation domain; • indicates the formation of hydrogen bonds between two adjacent nucleotides within of the HPH nucleic acid molecule; D comprises the nucleotide sequence that is complementary to the nucleotide sequence of O; D and O form base pairs greater than or equal to two with each other which are contiguous or non-contiguous; O and W are of greater length than or equal to 3 nucleotides; F, D, W and O together are of sufficient length to interact stably with the target sequence; each B and B 'independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond. 99.- A method for modulating the function of an objective sequence in a cell comprising the step of contacting said cell with a hairpin hybridizing nucleic acid (HPH) molecule under appropriate conditions to modulate the function of said target sequence, characterized in that said hairpin hybridizing nucleic acid molecule consists of the formula XXV: B B 'I F GONE W in which, each F, D, O, and W independently represents an oligonucleotide whose nucleotide sequence could be the same or different; could be of the same length or different length and could include in individually or in combination, internucleotide linkages that are selected from the group consisting of phosphodiester, phosphorothioate, 5'-thiophosphate, phosphorodithioate, and methylphosphonate; F and D independently form an RNAse H activation domain of greater than or equal to 4 nucleotides; • indicates the formation of hydrogen bonds between two adjacent nucleotides within the HPH nucleic acid molecule; D comprises the nucleotide sequence that is complementary to the nucleotide sequence of O; D and O form base pairs greater than or equal to two with each other which are contiguous or non-contiguous; O and W are of greater length than or equal to 3 nucleotides; F, D, W and O together are of sufficient length to interact stably with the target sequence; each B and B 'independently represent an end blocking structure which independently could be present or absent; and represents a chemical bond. 100.- A method for inhibiting the function of an objective sequence in a cell, characterized in that said target sequence is encoded by a c-raf gene, comprising the step of contacting said cell with a hybridizing nucleic acid molecule of hairpin (HPH) under conditions appropriate for the inhibition of said function of the target sequence, wherein said hairpin hybridizing nucleic acid molecule comprises any of the sequences that are selected from the group consisting of SEQ. ID. Nos. 1, 15, 17, 18, 19 and 20. 101.- A method to inhibit the function of a sequence target in a cell, characterized in that said target sequence is encoded by an IMPDH II gene, comprising the step of contacting said cell with a hairpin hybridizing nucleic acid (HPH) molecule under conditions appropriate for the inhibition of said function of the target sequence, wherein said hairpin hybridizing nucleic acid molecule comprises any of the sequences that are selected from the group consisting of SEQ. ID. Nos. 11, 13, 24, 25, 27 and 28.