Multiplexed in situ signal amplification with splint ligation extension (slx) probes

A multiplexed target detection method, the method comprising,

• (a) combining a plurality of single stranded nucleic acid constructs each with a predetermined number of binding sites for imaging oligonucleotides with a plurality of splint oligonucleotides, wherein the constructs each comprise:
• (i) a targeting domain, comprising at least one programmable unit with each unit comprising from a 5′ end to a 3′ end, a first orthogonal ligation adapter sequence, a target payload sequence comprising a predetermined number of binding sites, and a second orthogonal ligation adapter sequence;
• and
• (ii) a signaling domain, comprising at least two programmable units with each unit comprising from a 5′ end to a 3′ end, a first orthogonal ligation adapter sequence, a signaling payload sequence comprising a predetermined number of binding sites, and a second orthogonal ligation adapter sequence;
• and wherein the at least one splint oligonucleotide hybridizes to a complementary orthogonal ligation adapter sequence on a first unit and hybridizes to a complementary orthogonal ligation adapter sequence on a second unit to form a ligation junction to link the first programmable unit with the second programmable unit to form a first reaction mixture comprising a plurality of functional units;
• (b) combining the first reaction mixture produced in step (a) with a sample containing a plurality of nucleic acid targets, wherein the functional units in the first reaction mixture produced in step (a) each hybridize with a nucleic acid target through the targeting domain producing a second reaction mixture;
• (c) combining the second reaction mixture produced in step (b) with a plurality of imaging oligonucleotides, wherein the functional units in the second reaction mixture produced in step (b) each hybridize with an imaging oligonucleotide through the signaling domain;
• and
• (d) imaging the labeled functional units.

A method of assembling a single stranded nucleic acid probe construct, comprising:

• providing reaction mixture comprising:
• a targeting oligonucleotide, comprising in order a first orthogonal ligation adapter sequence at a first end, a targeting payload sequence that hybridizes to a target sequence in a nucleic acid of interest, and a second orthogonal ligation adapter sequence at a second end,
• one or more signal oligonucleotides, each comprising in order a first orthogonal ligation adapter sequence at a first end, a signaling payload sequence that hybridizes to an imaging oligonucleotide, and a second orthogonal ligation adapter sequence at a second end,
• a first splint oligonucleotide with a first domain that hybridizes to the second orthogonal ligation adapter sequence of the targeting oligonucleotide and a second domain that hybridizes to the first orthogonal ligation adapter sequence of a first signal oligonucleotide;
• permitting the first splint oligonucleotide to hybridize to the second orthogonal ligation adapter sequence of the targeting oligonucleotide and the first orthogonal ligation adapter sequence of the first signal oligonucleotide, thereby bringing the second end of the targeting oligonucleotide and the first end of the first signal oligo into close proximity;
• ligating the second end of the targeting oligonucleotide to the first end of the first signal oligonucleotide to provide a ligated probe precursor.

The method of claim 41, further comprising:

• (i) subjecting the amplified probe precursor to exonuclease digestion to provide a single stranded DNA probe construct;
• (ii) subjecting the amplified probe precursor to DNA nicking and gel purification to provide a single stranded DNA probe construct;
• or
• (iii) performing circle-to-circle DNA amplification to provide a single stranded DNA probe construct.