Abstract: The present disclosure in some aspects relates to methods and compositions for detecting and quantifying multiple analytes present in a biological sample by rolling circle amplification (RCA). In some aspects, the methods and compositions provided herein address issues associated with control of signal spot size and intensity for RCA products (RCPs) in a sample. In some aspects, provided herein are methods and compositions (e.g., probes and/or reaction mixtures) for slowing down RCA and/or decreasing the size of RCA products. In some embodiments, nucleotides or nucleotide analogs having a hydrophobic group (e.g., a hydrophobic modification on the base) are included in nucleotides for RCA and incorporated into the RCP product, leading to reduction of RCP size without the need of crosslinking an incorporated hydrophobic nucleotide or nucleotide analog to the RCP itself or another molecule.

Abstract: The present disclosure in some aspects relates to methods and compositions for accurately detecting and quantifying multiple analytes present in a biological sample. In some aspects, the methods and compositions provided herein address one or more issues associated with the stability and/or size of nucleic acid structures such as rolling circle amplification products in the biological sample.

Abstract: The present disclosure in some aspects relate to a direct RNA (dRNA) detection approach incorporating the use of circularizable probes (e.g., padlock probes) having asymmetric arms, rolling circle amplification of the ligated circularizable probes, and in situ detection (e.g., using hybridization-based in situ sequencing) for multiplexed spatial analysis of nucleic acid sequences (e.g., short sequences such as SNPs and point mutations) in a biological sample. In some aspects, compositions and methods disclosed herein improve detection specificity, reduce false positive signal detection, and/or maintain or improve detection efficiency.

Abstract: The present disclosure in some aspects relates to methods and compositions for accurately detecting and quantifying multiple analytes present in a biological sample. In some aspects, the methods and compositions provided herein address one or more issues associated with the stability and/or size of nucleic acid structures, such as RCPs, in the biological sample without the use of exogenously added oligonucleotide compaction probes. In some embodiments, provided herein are methods involving the use of self-hybridizing hybridizing regions for compacting and/or stabilizing nucleic acid concatemers (e.g., RCPs). In some embodiments, dynamic inter-strand annealing between tandem units of an RCP is used for compaction and/or stabilization. In some embodiments, short palindromic regions in an RCP are used for compaction and/or stabilization.

Abstract: The present disclosure in some aspects relates to methods and compositions for accurately detecting and quantifying multiple analytes present in a biological sample. In some aspects, the methods and compositions provided herein address issues associated with the heterogeneity of analyte abundance (e.g., gene expression levels) and variations among reactions at different locations of a sample (e.g., amplification reaction starting earlier at one location than another location). In some aspects, a method disclosed herein provides a tighter distribution of signal spot size and intensity in a sample, as compared to methods that result in a wide and heterogeneous size and intensity distribution of signal spots.

Abstract: The present disclosure relates in some aspects to methods and compositions for analysis of a target nucleic acid, such as in situ detection of a region of interest in a polynucleotide in a tissue sample. In some embodiments, provided herein are templated ligation probes (e.g., RNA-templated ligation probes) and selector probes for generation of a circularized ligated probe comprising an insertion sequence of a selector probe, wherein the circularized ligated probe is amplified in a rolling circle amplification reaction to generate a product that is detected in the sample.

Abstract: The present disclosure in some aspects relates to methods and compositions for accurately detecting and quantifying multiple analytes present in a biological sample. In some aspects, the methods and compositions provided herein allow for detection of a target sequence by rolling circle amplification without requiring a ligation step and without sacrificing specificity (e.g., rolling circle amplification occurs only for circular probes and/or hairpin molecules specifically hybridized to a target sequence).

Abstract: The present disclosure relates in some aspects to methods for analyzing a target nucleic acid in a biological sample. In some aspects, provided herein are methods and compositions for detecting a region of interest in a target nucleic acid, wherein hybridization between an interrogatory region of a probe and a region of interest of the target nucleic acid is blocked by a blocking strand unless the interrogatory region is complementary to the region of interest. In some aspects, the methods provided herein increase specificity of detecting a region of interest in a target nucleic acid (e.g., a SNP in an RNA molecule). In some aspects, the presence, amount, and/or identity of a region of interest in a target nucleic acid is analyzed in situ. Also provided are polynucleotides, sets of polynucleotides, compositions, and kits for use in accordance with the methods, for example for RNA-targeting padlock probe-mediated SNP detection.

Abstract: The present disclosure relates in some aspects to methods for analyzing a target nucleic acid in a biological sample. In some aspects, provided herein are methods and compositions for detecting a region of interest in a target nucleic acid.