Detection of oligonucleotides by dual hybridization

A method of detecting a VEGF aptamer having the structure:

• PEG-linker-CfGmGmArArUfCfAmGmUfGmAmAm
• (SEQ ID NO:1)
• UfGmCfUfUfAmUfAmCfAmUfCfCfGm 3′-3′-Td,
• in a biological sample, comprising:
• (a) contacting the sample with a first polynucleotide that is complementary to a first portion of the VEGF aptamer to allow hybridization of the first polynucleotide with the VEGF aptamer, the first polynucleotide being joined to a tag and having the sequence:
• 5′
• AAAACGGATGTATAAGCA-3′;
• (SEQ ID NO:3)
• (b) contacting the sample with a second polynucleotide comprising the sequence TTCACTGATTCCG (SEQ ID NO:
• 5), that is complementary to an adjacent and non-overlapping portion of the VEGF aptamer to allow hybridization of the second polynucleotide with the VEGF aptamer, the second polynucleotide further comprising an amplifiable sequence that is substantially non-complementary to the VEGF aptamer;
• (c) ligating the first and the second polynucleotides together, wherein the first and second polynucleotides are hybridized adjacently to the VEGF aptamer, to form a ligated hybrid complex;
• (d) separating the ligated hybrid complex from the rest of the sample using the tag of the first polynucleotide;
• and
• (e) detecting the amplifiable sequence of the second polynucleotide by a polymerase chain reaction amplification using a fluorescent probe having the structure:
• 6FAM-AAG GAA CTC TTC TGG AAT GA-MGBNFQ (SEQ ID NO:
• 9), wherein the amplifiable sequence is detected when the VEGF aptamer is present in the biological sample,
• thereby detecting the VEGF aptamer in the biological sample.

A kit for detecting a target oligonucleotide in a sample, comprising:

• (a) a first polynucleotide that is complementary to a first portion of the target oligonucleotide, so as to allow hybridization of the first polynucleotide with the target oligonucleotide, said first polynucleotide is joined to a tag;
• (b) a second polynucleotide that is complementary to an adjacent and non-overlapping portion of the target oligonucleotide so as to allow hybridization of the second polynucleotide with the target oligonucleotide, said second polynucleotide comprises an amplifiable sequence that is substantially non-complementary to the target oligonucleotide;
• and
• (c) a fluorescent probe comprising a nucleic acid complementary to at least a portion of the amplifiable sequence.

A method of quantifying a target oligonucleotide in a sample, comprising the steps of:

• (a) contacting the sample with a capture primer that is complementary to a first portion of the target oligonucleotide, so as to allow hybridization of the capture primer with the target oligonucleotide, said capture primer comprises a first amplifiable sequence that is substantially non-complementary to the target oligonucleotide, said capture primer is joined to a tag;
• (b) contacting the sample with a detection primer that is complementary to an adjacent and non-overlapping portion of the target oligonucleotide so as to allow hybridization of the detection primer with the target oligonucleotide thereby forming a hybrid complex, said detection primer comprises a second amplifiable sequence that is substantially non-complementary to the target oligonucleotide;
• (c) measuring the quantity of the first amplifiable sequence by real-time-PCR;
• and
• (d) measuring the quantity of the second amplifiable sequence by real-time-PCR,
• whereby, measuring the amount of the first amplifiable sequence indicates the amount of capture primer present in the sample and measuring the amount of the second amplifiable sequence indicates the amount of target oligonucleotide present in the sample.