The present disclosure describes compositions of matter comprising a ribonucleoprotein complex comprising a nucleic acid-guided nuclease and a guide RNA, and further comprising and a blocking nucleic acid molecule represented by Formula I, wherein Formula I in the 5′-to-3′ direction comprises: A-(B-L).sub.J-C-M-T-D; wherein A is 0-15 nucleotides in length; B is 4-12 nucleotides in length; L is 3-25 nucleotides in length; J is an integer between 1 and 10; C is 4-15 nucleotides in length; M is 1-25 nucleotides in length or is absent, wherein if M is absent then A-(B-L).sub.J-C and T-D are separate nucleic acid strands; T is 17-135 nucleotides in length and comprises at least 50% sequence complementarity to B and C; D is 0-10 nucleotides in length and comprises at least 50% sequence complementarity to A; and wherein the blocking nucleic acid molecule comprises a sequence complementary to a gRNA.

The present disclosure describes compositions of matter comprising a ribonucleoprotein complex comprising a nucleic acid-guided nuclease and a guide RNA, and further comprising and a blocking nucleic acid molecule represented by Formula I, wherein Formula I in the 5′-to-3′ direction comprises: A-(B-L).sub.J-C-M-T-D; wherein A is 0-15 nucleotides in length; B is 4-12 nucleotides in length; L is 3-25 nucleotides in length; J is an integer between 1 and 10; C is 4-15 nucleotides in length; M is 1-25 nucleotides in length or is absent, wherein if M is absent then A-(B-L).sub.J-C and T-D are separate nucleic acid strands; T is 17-135 nucleotides in length and comprises at least 50% sequence complementarity to B and C; D is 0-10 nucleotides in length and comprises at least 50% sequence complementarity to A; and wherein the blocking nucleic acid molecule comprises a sequence complementary to a gRNA.

The disclosure provides a method for detecting a target analyte in a biological sample including contacting the sample with one or more probe sets each comprising a primary probe and a linker, contacting the sample with an initiator sequence, contacting the sample with a plurality of fluorescent DNA hairpins, wherein the probe binds the target molecule, the linker connects the probe to the initiator sequence, and wherein the initiator sequence nucleates with the cognate hairpin and triggers self-assembly of tethered fluorescent amplification polymers, and detecting the target molecule by measuring fluorescent signal of the sample.

The disclosure provides a method for detecting a target analyte in a biological sample including contacting the sample with one or more probe sets each comprising a primary probe and a linker, contacting the sample with an initiator sequence, contacting the sample with a plurality of fluorescent DNA hairpins, wherein the probe binds the target molecule, the linker connects the probe to the initiator sequence, and wherein the initiator sequence nucleates with the cognate hairpin and triggers self-assembly of tethered fluorescent amplification polymers, and detecting the target molecule by measuring fluorescent signal of the sample.

The disclosure provides a method for detecting a target analyte in a biological sample including contacting the sample with one or more probe sets each comprising a primary probe and a linker, contacting the sample with an initiator sequence, contacting the sample with a plurality of fluorescent DNA hairpins, wherein the probe binds the target molecule, the linker connects the probe to the initiator sequence, and wherein the initiator sequence nucleates with the cognate hairpin and triggers self-assembly of tethered fluorescent amplification polymers, and detecting the target molecule by measuring fluorescent signal of the sample.

The technology provided herein relates to multiplex methods and kits for detecting different analytes in a sample in parallel by sequential signal-encoding of said analytes, as well as in vitro methods for screening, identifying and/or testing a substance and/or drug and in vitro methods for diagnosis of a disease, and an optical multiplexing system.

The technology provided herein relates to multiplex methods and kits for detecting different analytes in a sample in parallel by sequential signal-encoding of said analytes, as well as in vitro methods for screening, identifying and/or testing a substance and/or drug and in vitro methods for diagnosis of a disease, and an optical multiplexing system.

The technology provided herein relates to multiplex methods and kits for detecting different analytes in a sample in parallel by sequential signal-encoding of said analytes, as well as in vitro methods for screening, identifying and/or testing a substance and/or drug and in vitro methods for diagnosis of a disease, and an optical multiplexing system.

The present application provides methods for detecting a target nucleic acid molecule in a sample comprising contacting said sample with a ligatable probe comprising one or more parts and allowing said probe to hybridise to the target nucleic acid molecule, ligating any probe which has hybridised to the target nucleic acid molecule, amplifying the ligated probe, and detecting the amplification product, thereby to detect the target nucleic acid molecule, wherein said probes comprise at least one ribonucleotide at or near to a ligation site and/or wherein the probe or a probe part comprises an additional sequence 5′ to a target-specific binding site which is not hybridised to the target nucleic acid molecule upon hybridisation of the probe to the target nucleic acid molecule and forms a 5′ flap containing one or more nucleotides at its 3′ end that is cleaved prior to ligation, and methods of synthesising a DNA molecule with Phi29 DNA polymerase using a template nucleic acid molecule comprising at least one ribonucleotide. Probes for use in the detection methods are provided.

The present application provides methods for detecting a target nucleic acid molecule in a sample comprising contacting said sample with a ligatable probe comprising one or more parts and allowing said probe to hybridise to the target nucleic acid molecule, ligating any probe which has hybridised to the target nucleic acid molecule, amplifying the ligated probe, and detecting the amplification product, thereby to detect the target nucleic acid molecule, wherein said probes comprise at least one ribonucleotide at or near to a ligation site and/or wherein the probe or a probe part comprises an additional sequence 5′ to a target-specific binding site which is not hybridised to the target nucleic acid molecule upon hybridisation of the probe to the target nucleic acid molecule and forms a 5′ flap containing one or more nucleotides at its 3′ end that is cleaved prior to ligation, and methods of synthesising a DNA molecule with Phi29 DNA polymerase using a template nucleic acid molecule comprising at least one ribonucleotide. Probes for use in the detection methods are provided.