Photocleavable oligonucleotide and uses thereof
View Patent ↗This invention relates to methods and compositions of oligonucleotide constructs having a photocleavable linker. Specifically, provided herein are methods and compositions utilizing a photocleavable linker, which when exposed to light modulates the expression of genes.
1. A photoactive oligonucleotide conjugate having the structure:
X-Y-Z
wherein the X component is an antisense oligonucleotide of between 15 and 50 nucleotides in length; the Z component is a sense oligonucleotide that includes a sequence complementary to a first portion of at least 10 consecutive nucleotides of said antisense oligonucleotide; and the Y component is a photocleavable linker covalently attached therebetween, wherein the antisense oligonucleotide has a second portion of at least 5 consecutive nucleotides that is not complementary to the sense oligonucleotide and/or the antisense oligonucleotide is at least 5 nucleotides longer than the sense oligonucleotide, and wherein cleavage of said photocleavable linker results in a lower melting temperature (T m ) of the antisense oligonucleotide to the sense oligonucleotide of between about 20 to 50° C. of the cleaved conjugate relative to the uncleaved conjugate.
2. The conjugate of claim 1 , wherein the antisense oligonucleotide is between 15 and 20 nucleotides in length.
3. The conjugate of claim 1 , wherein the 5′ end of the antisense oligonucleotide and the 3′ end of the sense oligonucleotide each contain a region that is not complementary to the opposite strand.
4. The conjugate of claim 3 , wherein the region is no less than 5 nucleotides in length.
5. The conjugate of claim 3 , wherein the antisense oligonucleotide is a DNA, a peptide nucleic acid (PNA), a negatively charged peptide nucleic acid (ncPNA), a phosphorothioate DNA (PS-DNA), a phosphorodiamidate morpholino oligonucleotide (PMO), or a locked nucleic acid (LNA).
6. The conjugate of claim 1 , wherein the photocleavable linker is capable of reacting with thiol and amine functionalities.
7. The conjugate of claim 1 , wherein the photocleavable linker is 1-(5-(N-maleimidomethyl)-2-nitrophenyl)ethanol N-hydroxysuccinimide ester.
8. The conjugate of claim 1 , wherein the photocleavable linker comprises a maleimido moiety.
9. The conjugate of claim 1 , wherein the photocleavable linker comprises a N-hydroxysuccinimide ester moiety.
10. The conjugate of claim 1 , wherein the sense oligonucleotide comprises a DNA, a peptide nucleic acid (PNA), a negatively charged peptide nucleic acid (ncPNA), a phosphorothioate DNA (PS-DNA), a phosphorodiamidate morpholino oligonucleotide (PMO), a locked nucleic acid (LNA), or 2′-O-methyl RNA (2′-OMe RNA).
11. A composition for reducing expression of a gene of interest in a subject comprising a photocleavable conjugate, wherein the conjugate comprises an antisense oligodeoxynucleotide (asODN) of between 15 and 50 nucleotides in length with at least 15 consecutive nucleotides which are no less than 85% complementary to the gene of interest, a sense strand complementary to a portion of at least 10 consecutive nucleotides of said asODN, and a photocleavable linker attached therebetween; wherein cleaving the photocleavable linker of the conjugate activates the asODN to react with the gene of interest, wherein cleavage of said photocleavable linker results in a lower melting temperature (T m ) of the antisense oligonucleotide to the sense oligonucleotide of between about 20 to 50° C. of the cleaved conjugate relative to the uncleaved conjugate, and wherein hybridization of the cleaved antisense oligonucleotide with the gene of interest is more energetically preferable than reassociation with the sense strand.
12. The composition of claim 11 wherein the sense oligonucleotide comprises a DNA, a peptide nucleic acid (PNA), a negatively charged peptide nucleic acid (ncPNA), a phosphorothioate DNA (PS-DNA), a phosphorodiamidate morpholino oligonucleotide (PMO), or a locked nucleic acid (LNA), or 2′-O-methyl RNA (2′-OMe RNA).