RNA-DNA hybrid molecules of nucleic acid

• US 5,708,154 A
• Filed: 08/16/1991
• Issued: 01/13/1998
• Est. Priority Date: 02/24/1989
• Status: Expired due to Term

• Current Assignee: City of Hope
• Original Assignee: City of Hope
• Inventors: Smith, Steven S., Kaplan, Bruce E.
• Primary Examiner(s): Wilson, James O.
• Application Number:
  US07/745,922
• Time in Patent Office: 2,342 Days
• Field of Search: 536/27, 536/28, 536/29, 536/23.1, 536/25.3, 536/25.32, 536/25.34, 536/25.1, 435/6, 435/28, 435/7.5, 435/91.1, 435/91.51
• US Class Current: 536/23.1
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2525/121 incorporating both deoxyribonucleotides and ribonucleotides
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides

Abstract

Molecules having an RNA block linked to a shorter DNA block is described. The RNA and DNA blocks are complementary to accommodate formation of fold-back molecules having a 3'"'"' hydroxyl on the DNA block and an overhanging RNA strand at the end of a short DNA-RNA hybrid.

Claims

1. A modular nanostructure comprising

(i) a thirty base RNA sequence;

(ii) a ten base DNA sequence

said DNA sequence being hybridized to the 10 bases adjacent the 3'"'"' terminus of said RNA sequence to provide an unhybridized 20 base sequence at the 5'"'"' terminus of said RNA sequence, and

(iii) a five dT moiety tether connecting the 3'"'"' terminal nucleotide of said RNA sequence to the 5'"'"' terminal nucleotide of said DNA sequence.

2. A modular nanostructure as defined by claim 1 in which said tether has a fluorescent label.

3. A modular nanostructure as defined by claim 1 in which the dT moiety is hydrophilic.

4. A modular nanostructure as defined by claim 1 in which said RNA sequence includes an enzyme inhibiting moiety.

5. A modular nanostructure as defined by claim 4 in which said enzyme inhibitor is 1,4 anhydro-D-ribitol or 3'"'"' deoxyadenosine.

12. A modular nanostructure as defined by claim 1 in which said tether has a fluorescein isothiocyanate label.

13. A modular nanostructure as defined by claim 1 in which said RNA sequence has a single substituent, said single substituent being 1,4 anhydro-D-ribitol or 3'"'"' deoxyadenosine.

6. A molecule having the schematic formula:

##STR3## in which R is an RNA sequence of x nucleotides, D is a DNA sequence of y nucleotides, Z is an organic moiety linking the 3'"'"' terminal nucleotide of R with the 5'"'"' terminal nucleotide of D, and the value of x is more than the value of y

a 3'"'"' terminal portion of R adjacent Z being complementary to a 5'"'"' terminal portion of D adjacent Z wherein said 3'"'"' and 5'"'"' terminal portions of R and D hybridize to provide an RNA-DNA hybrid sequence, and an unhybridized 5'"'"' terminal portion of R.

7. A molecule as defined by claim 6 in which Z comprises dT residues.

8. A molecule as defined by claim 6 in which Z is labelled.

9. A molecule as defined by claim 6 in which R or D are substituted by an enzyme inhibiting moiety.

10. A molecule as defined by claim 6 in which R is substituted by 1,4 anhydro-D-ribitol or 3'"'"' deoxyadenosine.

15. A molecule as defined by claim 6 in which Z consists of dT residues.

16. A molecule as defined by claim 6 in which Z consists of dT residues and in which Z has a fluorescein isothiocyanate label.

17. A molecule as defined by claim 6 in which either R or D has a single substituent and where said single substituent is 1,4 anhydro-D-ribitol or 3'"'"' deoxyadenosine.

18. A molecule as defined by claim 6 in which Z consists of five dT residues, R is a thirty base RNA sequence, D is a ten base DNA sequence,

wherein D is hybridized to the ten bases adjacent the 3'"'"' terminus of R.

19. A molecule as defined by claim 6 in which R or D has a single enzyme inhibiting moiety attached thereto and in which said enzyme inhibiting moiety is 1,4 anhydro-D-ribitol or 3'"'"' deoxyadenosine.

11. A modular nanostructure consisting of

(i) a thirty base RNA sequence;

(ii) a ten base DNA sequence said DNA sequence being hybridized to the 10 bases adjacent the 3'"'"' terminus of said RNA sequence to provide an unhybridized 20 base sequence at the 5'"'"' terminus of said RNA sequence, and

(iii) a five dT moiety tether connecting the 3'"'"' terminal nucleotide of said RNA sequence to the 5'"'"' terminal nucleotide of said DNA sequence.

14. A molecule having the schematic formula:

##STR4## in which R is an RNA sequence of x nucleotides, D is a DNA sequence of y nucleotides, Z is a sequence of dT residues and the value of X is greater than the value of y and in which a 3'"'"' terminal portion of R adjacent Z is complementary to a 5'"'"' terminal portion of D adjacent Z;

wherein said 3'"'"' and 5'"'"' terminal portions of R and D are hybridized to provide an RNA-DNA hybrid sequence, and an unhybridized 5'"'"' terminal portion of R.

20. A molecule having the formula:

##STR5## in which R is an RNA sequence of x nucleotides, D is a DNA sequence initially of y nucleotides, Z is an organic moiety linking the 3'"'"' terminal nucleotide of R with the 5'"'"' terminal nucleotide of D, and the value of x is more than the value of y;

a 3'"'"' terminal portion of R adjacent Z being complementary to a 5'"'"' terminal portion of D adjacent Z wherein said 3'"'"' and 5'"'"' terminal portions of R and D hybridize to provide an RNA-DNA hybrid sequence, and an unhybridized 5'"'"' terminal portion of R; and

in which the 3'"'"' terminal position of D has been extended by reverse transcriptase in the presence of a biotinylated deoxynucleotide precursor so that D is a DNA sequence of x nucleotides and in which the D sequence includes biotinylated nucleotides in the region corresponding to the previously unhybridized portion of the R sequence.