Method of Detection of Nucleic Acids with a Specific Sequence Composition

Abstract

This invention is a novel method for detecting and localizing specific nucleic acid sequences in a sample with a high degree of sensitivity and specificity. The method and novel compositions used in the method involve the use of Probe Nucleic Acids, the production of nucleic acid binding regions and the use of nucleic acid Target Binding Assemblies to detect and localize specific Target Nucleic Acids. The detection and localization of the Target Nucleic Acid is accomplished even in the presence of nucleic acids which have similar sequences. The method provides for a high degree of amplification of the signal produced by each specific binding event. In particular, methods and compositions are presented for the detection of HIV and HPV nucleic acid in samples. These methods and compositions find use in diagnosis of disease, genetic monitoring, forensics, and analysis of nucleic acid mixtures. Some of the novel compositions used in the detection method are useful in preventing or treating pathogenic conditions.

1 . An isolated target binding assembly (TBA) comprising a first and a second nucleic acid recognition unit, wherein:
 said first nucleic acid recognition unit is capable of binding with specificity to a first target nucleotide sequence contained within a target polynucleotide;   said second nucleic acid recognition unit is capable of binding with specificity to a second target nucleotide sequence contained within a target polynucleotide; and   said nucleic acid recognition units are polypeptides and are the same or different.   
     
     
         2 . The TBA of  claim 1 , wherein each said target nucleotide sequence includes at least 7 contiguous nucleotides. 
     
     
         3 . The TBA of  claim 1 , wherein said first nucleic acid recognition unit is capable of binding said first target nucleotide sequence at the same time that said second nucleic acid recognition unit is bound to said second target nucleotide sequence, when said first and second target nucleotide sequences:
 are both contained within the same target polynucleotide; and   are separated from one another by intervening nucleotide(s).   
     
     
         4 . The TBA of  claim 3 , wherein each said target nucleotide sequence includes at least 7 contiguous nucleotides. 
     
     
         5 . The TBA of  claim 2 , wherein said first and second nucleic acid recognition units are DNA-binding polypeptides. 
     
     
         6 . The TBA of  claim 2 , wherein said first and second nucleic acid recognition units are RNA-binding polypeptides. 
     
     
         7 . The TBA of  claim 3 , wherein said first and second nucleic acid recognition units are DNA-binding polypeptides. 
     
     
         8 . The TBA of  claim 3 , wherein said first and second nucleic acid recognition units are RNA-binding polypeptides. 
     
     
         9 . The TBA of  claim 4 , wherein said first and second nucleic acid recognition units are DNA-binding polypeptides that bind reversibly and non-covalently to double-stranded DNA. 
     
     
         10 . The TBA of  claim 9 , wherein said first and second nucleic acid recognition units, when considered separately, each have a binding affinity K d  for their respective target nucleotide sequence that is numerically greater than 10 −6  M. 
     
     
         11 . A composition comprising the TBA of  claim 1  bound to a target polynucleotide comprising said first target nucleotide sequence and said second target nucleotide sequence, whereby transcription of a gene contained within the target polynucleotide is hindered. 
     
     
         12 . The TBA according to  claim 1 , wherein said TBA comprises a sequence selected from the group consisting of SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, and SEQ ID NO: 116. 
     
     
         13 . The TBA according to  claim 1 , wherein said TBA comprises a sequence selected from the group consisting of:
 A. I+II+III;   B. IV+V+III; and   C. IV+III;   
       wherein
 I indicates any of SEQ ID NOS. 85-92; 
 II indicates Met Ser, linked to any of SEQ ID NOS 104-106, each of which is linked to SEQ ID NO. 99; 
 III indicates SEQ ID NO. 100 linked to any of SEQ ID NOS. 75-84 or 94-98; SEQ ID NO. 101 linked to either SEQ ID NO. 74 or SEQ ID NO. 93; or SEQ ID NO. 102 linked to SEQ ID NO. 74 or SEQ ID NO. 93; or any of SEQ ID NO. 72, 103, 73, or 63-71; 
 IV indicates any of SEQ ID NOS. 104-106; and 
 V indicates SEQ ID NO. 99. 
 
     
     
         14 . The TBA according to  claim 13 , wherein said TBA comprises a sequence selected from the group consisting of SEQ ID NOS:104-106. 
     
     
         15 . The TBA according to  claim 13 , wherein said TBA comprises a sequence selected from Group B or Group C. 
     
     
         16 . A method of treatment or prophylaxis comprising administering a TBA according to  claim 1 , or a polynucleotide encoding said TBA, to a patient in need thereof. 
     
     
         17 . The TBA according to  claim 1 , wherein at least one of said first and a second nucleic acid recognition units is a binding domain selected from NF-kB, papillomavirus E2 protein, transcription factor SP1, an inactive restriction enzyme, or an antibody. 
     
     
         18 . The TBA according to  claim 1 , wherein said TBA is HIV-Detect I, HIV-Detect II, HIV Detect-III, HIV-Detect IV, HPV-Detect I, HPV-Detect II, HPV-Detect III, or HPV-Detect IV. 
     
     
         19 . The TBA according to  claim 1 , wherein said TBA is HIV-Lock.