Telomere-encoding synthetic DNA nanocircles, and their use for the elongation of telomere repeats
AssigneeThe Board of Trustees of the Leland Stanford Junior University
InventorEric T. Kool
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Abstract
Telomere-encoding nucleic acid nanocircles, methods for their preparation, and methods for their use are disclosed. The nanocircles can be constructed containing multiple repeats of the complement of telomere repeat sequences. The telomere-encoding nanocircles are useful for extending telomeres both in vitro and in vivo, for treating macular degeneration, the effects of skin aging, liver degeneration, and cancer. The nanocircles are further useful for treating cell cultures to produce long-lived non-cancerous cell populations. This use has wide applicability in scientific research, tissue engineering, and transplantation.
Claims26
A nucleic acid nanocircle comprising at least 2 repeats of a telomere repeat sequence, wherein the nanocircle is up to about 2004 nucleotides in length.
A nucleic acid nanocircle comprising at least 2 repeats of 5′-CCCTAA-3′ (SEQ ID NO:3), 5′-CCCUAA-3′ (SEQ ID NO:4), 5′-TTAGGG-3′ (SEQ ID NO:63), or 5′-UUAGGG-3′ (SEQ ID NO:64), wherein the nanocircle is up to about 2004 nucleotides in length.
A nucleic acid nanocircle comprising at least 2 repeats of a telomere repeat sequence, wherein at least about 50% of the sequence of the nanocircle is an integral number of repeats of the telomere repeat sequence.
A nucleic acid nanocircle comprising at least 2 repeats of 5′-CCCTAA-3′ (SEQ ID NO:3), at least 2 repeats of 5′-CCCUAA-3′ (SEQ ID NO:4), at least 2 repeats of 5′-TTAGGG-3′ (SEQ ID NO:63), or at least 2 repeats of 5′-UUAGGG-3′ (SEQ ID NO:64), wherein:
- at least about 50% of the sequence of the nanocircle is an integral number of repeats of 5′-CCCTAA-3′ (SEQ ID NO:3), 5′-CCCUAA-3′ (SEQ ID NO:4);
- 5′-TTAGGG-3′ (SEQ ID NO:63), or 5′-UUAGGG-3′ (SEQ ID NO:64).
A nucleic acid nanocircle consisting essentially of at least two repeats of a telomere repeat sequence.
A nucleic acid nanocircle consisting essentially of at least two repeats of 5′-CCCTAA-3′ (SEQ ID NO:3), 5′-CCCUAA-3′ (SEQ ID NO:4), 5′-TTAGGG-3′ (SEQ ID NO:63), or 5′-UUAGGG-3′ (SEQ ID NO:64).
A nucleic acid nanocircle consisting of at least two repeats of a telomere repeat sequence.
A method to prepare a nucleic acid nanocircle consisting of an integral number of repeats of a nucleic acid sequence, the method comprising:
- obtaining a protected linear precursor nucleic acid molecule having:
- a first protecting group attached to one or more nucleotides at or near the 5′ end of the linear precursor nucleic acid molecule, and
- a second protecting group attached to one or more nucleotides at or near the 3′ end of the linear precursor nucleic acid molecule, and
- at least one third protecting group attached to the linear precursor nucleic acid molecule between the 5′ end and the 3′ end of the linear precursor nucleic acid molecule;
- treating the protected linear precursor nucleic acid molecule under conditions suitable to remove the first protecting group and the second protecting group from the protected linear precursor nucleic acid molecule to produce a partially deprotected linear precursor nucleic acid molecule;
- contacting the partially deprotected linear precursor nucleic acid molecule with a splint nucleic acid molecule to form a linear/splint complex, wherein the splint nucleic acid molecule binds to both the 5′ end and to the 3′ end of the partially deprotected linear precursor nucleic acid molecule;
- contacting the linear/splint complex with a ligation agent to produce a partially protected nucleic acid nanocircle;
- and
- treating the partially protected nucleic acid nanocircle under conditions suitable to remove the third protecting group to produce the nucleic acid nanocircle.
A method to extend the length of a telomere in vitro, the method comprising:
- selecting a telomere primer;
- and
- contacting the telomere primer with a nucleic acid nanocircle, a polymerase, and deoxyribonucleoside triphosphates under conditions suitable for polymerase activity.
A method to extend the length of a telomere in vivo, the method comprising:
- selecting a cell comprising a telomere;
- and
- contacting the cell with a nucleic acid nanocircle under conditions suitable for uptake of the nanocircle by the cell.
A method for treating macular degeneration, the method comprising:
- selecting a mammal comprising an eye that is experiencing macular degeneration;
- and
- contacting the eye and a nucleic acid nanocircle under conditions suitable for uptake of the nanocircle by the eye.
A method for treating the effects of skin aging, the method comprising:
- selecting a mammal comprising skin that is experiencing the effects of skin aging;
- and
- contacting the skin and a nucleic acid nanocircle under conditions suitable for uptake of the nanocircle by the skin.
A method for treating the effects of liver degeneration, the method comprising:
- selecting a mammal comprising a liver that is experiencing liver degeneration;
- and
- administering to the mammal a nucleic acid nanocircle under conditions suitable for uptake of the nanocircle by the liver.
A method for treating cancer in a mammal, the method comprising:
- selecting a mammal diagnosed with cancer;
- and
- administering to the mammal a nucleic acid nanocircle under conditions suitable for uptake of the nanocircle by cancer cells.
Dependencies are inferred from the claim text.