METHOD OF DETECTING TARGET NUCLEIC ACID MOLECULES
20230313290 · 2023-10-05
Assignee
Inventors
Cpc classification
C12Q2525/179
CHEMISTRY; METALLURGY
C12Q2525/179
CHEMISTRY; METALLURGY
International classification
Abstract
The present application provides methods for detecting a nucleic acid molecule involving the use of a signal code sequence which corresponds to said nucleic acid molecule and a plurality of labelled detection probes which yield signals which make up the signal code sequence. In particular, the invention provides a sequential barcoding and decoding scheme which utilises a sequencing-by-hybridisation (SBH) strategy to sequence and decode a nucleotide barcode sequence, and to differentiate the nucleotide barcode sequence from other nucleotide barcode sequences. In an extension of the method, the application also provides a new coding scheme for providing a target nucleic acid with a detectable “colour” (or similar signal)-based code.
Claims
1-50. (canceled)
51. A system for detecting a nucleic acid barcode sequence, the system comprising: (a) a padlock probe comprising target binding sites capable of hybridizing to a target sequence in a target nucleic acid molecule, wherein the padlock probe comprises a nucleotide barcode sequence that identifies the padlock probe, and wherein the nucleotide barcode sequence comprises a first domain sequence and a second domain sequence, wherein a portion of the first domain sequence overlaps with some but not all of the second domain sequence; (b) a universal pool of reporter probes, wherein the universal pool of reporter probes comprises at least two different species of reporter probes, wherein the first species of reporter probe comprises a first optically detectable moiety, and the second species of reporter probe comprises a second optically detectable moiety; (c) a first detection probe, wherein the first detection probe comprises (i) a recognition sequence comprising the first domain sequence, and (ii) a first overhang sequence comprising a reporter probe binding site complementary to a reporter probe of the universal pool of reporter probes; (c) a second detection probe, wherein the second detection probe comprises (i) a recognition sequence complementary the second domain sequence, and (ii) a second overhang sequence comprising a reporter probe binding site complementary to a reporter probe of the universal pool of reporter probes; wherein the second detection probe is capable of initiating a strand displacement reaction to displace the first detection probe hybridized to the first domain.
52. The system of claim 51, wherein the target sequence is in a native genomic DNA or in a naturally occurring RNA molecule, or in a cDNA, or in an amplification product generated from any of the foregoing nucleic acid molecules.
53. The system of claim 51, wherein the target nucleic acid molecule is linked to an antibody.
54. The system of claim 51, wherein each of the first optically detectable moiety and the second optically detectable moiety is a fluorophore.
55. The system of claim 54, wherein the series of signal codes is a unique series of fluorescent colors.
56. The system of claim 51, for detecting multiple different nucleic acid molecules present in the sample, wherein the system comprises a specific padlock probe with a different nucleotide barcode sequence assigned a different series of signal codes for each different nucleic acid molecule.
57. The system of claim 51, wherein the number of different species of reporter probes in the universal pool of reporter probes is four.
58. The system of claim 55, wherein the number of different barcode sequences is 50 or more.
59. The system of claim 51, wherein the biological sample is a cell or a tissue sample on a solid substrate.
60. The system of claim 51, wherein the first overhang sequence and the second overhang sequence are the same.
61. The system of claim 51, wherein the first overhang sequence and the second overhang sequence are different.
62. The system of claim 51, wherein the first domain is 5′ to the second domain in the nucleotide barcode sequence.
63. The system of claim 51, wherein the first domain is 3′ to the second domain in the nucleotide barcode sequence.
64. The system of claim 51, wherein the first domain and the second domain each comprises a first subunit and a second subunit, and wherein the second subunit from the first domain fully overlaps with the first subunit of the second domain.
65. The system of claim 51, wherein the nucleotide barcode sequence comprises: (i) a first common region adjacent to the first subunit of the first domain, wherein the recognition sequence of the first detection probe comprises a sequence which is complementary to the first common region; and (ii) a second common region adjacent to the second subunit of the second domain, wherein the recognition sequence of the second detection probe comprises a sequence which is complementary to the second common region.
66. The system of claim 65, wherein the first detection probe and the second detection probe each comprise a displacer toehold overhang region.
67. The system of claim 51, wherein the first domain and the second domain each comprises a unique subunit.
68. The system of claim 51, wherein each domain comprises a unique subunit.
69. The system of claim 51, wherein the system comprises reagents for circularizing said padlock probe; and reagents for performing rolling circle amplification of said circularized padlock probe to generate a rolling circle product (RCP), the RCP comprising multiple complementary copies of the nucleotide barcode sequence.
Description
[0209] The invention will now be described in more detail in the following non-limiting Examples. In addition, a set of drawings is presented in which:
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EXAMPLES
Example 1 - Sequencing by Hybridization to Detect a Discontinuous Barcode Sequence
General Principle
[0228] A first detection probe hybridizes to and decodes a first barcode subunit. A second detection probe then hybridizes in such a way that it displaces a part of the first detection probe. Each probe in the set of second probes used to interrogate the second barcode subunit comprises a common toehold region complementary to a sequence adjacent to the barcode subunit, to which it can hybridize very rapidly. The detection probe that is complementary to the second barcode subunit can then also hybridize to the overlapping part of the probe to which the first probe has bound and in this way can displace the first probe very efficiently. There is virtually no need for washing or formamide treatment which makes the method faster, cheaper and easier. The first probe, even though it still has the some affinity to the target is in minority (in much lower concentration) and hence, has a very little chance to hybridize and displace the second probe again and will eventually dissociate. The affinity of the second set of probes could even be increased by giving it one or two bases longer hybridization sequences in the toehold region.
[0229] The scheme works with barcode subunits that are individual bases (A, G, C and T) or duplets (AA, CC, GG, TT) or a mix of bases (AT, CG, TA, GC) or - preferably- triplets where at least two bases within the triplets are unique (two bases mismatch discrimination) to ensure high specificity.
[0230] Padlock probes can be provided with a barcode sequences with single base barcode subunits flanked by common spacer sequences in order to prove the principle. In this example a padlock probe against Actb was produced, with a single base difference in the middle of a common barcode sequence. RCA products were generated in vitro or in situ, inside fixed mouse brain tissue sections and were then interrogated with a set of probes targeting that single base barcode position. The barcodes were effectively decoded by a sequencing by hybridisation reaction using the detection probes.
Methods
RCA Generation in Vitro
[0231] Initial circular templates for rolling circle amplification were generated by performing a padlock probe ligation reaction templated by a single-stranded DNA template. The ligation of padlock probes was performed with a mix composed of 10 nM padlock probe (PO4-AGCCTCGCCTTTGCCTTTTCTACGATTTTACCAGTGGCTTTTGCGTCTATTTAGTGGAGC CtaacgctagaCTATCTTTCGCCCCGCGAGCACAG, SEQ ID NO: 1), 30 nM template (GGCAAAGGCGAGGCTCTGTGCTCGCGGGGC, SEQ ID NO: 2), T4 ligase reaction buffer (66 mM Tris-HCl (pH 7.5), 10 mM DTT, 10 mM MgCl2), 0.2 .Math.g/.Math.l BSA, 0.68 mM ATP) and 1 U T4 ligase in a final volume of 50 .Math.l. The mixture was incubated at 37° C. for 15 min followed by 65° C. for 2 min. Resulting circles from this reaction were diluted to 10 pM in mQ H.sub.2O and thereafter amplified by a target-primed RCA reaction, for which a mixture comprising 5 pM ligated circles, 0.2 .Math.g/.Math.l BSA, ϕ29 polymerase reaction buffer (Thermo Fisher), 125 .Math.M dNTPs and 0.4 U/.Math.l ϕ29 polymerase (Thermo Fisher) was used to amplify the abovementioned dilution of circles. The RCA reaction was incubated at 37° C. for 3 h and 65° C. for 2 min for heat inactivation.
[0232] 10.Math.L of the resulting RCA reaction was then applied to positively charged microscope slides (Superfrost, Thermo Fisher), covered with a 20x20mm coverslip and incubated at room temperature for 15 min to allow RCA products to bind to the positively charged surface of the microscope slide. The coverslip was then removed, the slide washed in PBS twice and the RCA products were then subjected to sequencing by hybridization mix.
RCA Generation in Situ, Inside Fixated Mouse Brain Tissue Sections:
[0233] Mouse strain C57BL/6 at 30 days age (P30) was euthanized and the olfactory bulb was dissected via cryosectioning. Cryosectioning was performed on ThermoFisher cryostat, at 10 .Math.m thickness. Sections were then adhered onto ThermoFisher Superfrost glass slides and stored at -70C until processing.
[0234] Slides were first removed from -70° C. storage and left to thaw at room temperature (RT) for 5 min. Fixation step was conducted by incubating slides in 3.7% PFA in 1x DEPC-PBS (KI Substrat, MIK3125-1L) at RT for 5 min. The slides were then washed in 1x DEPC-PBS for 1 min to ensure PFA removal, before permeabilization in 0.1 M HCl (KI Substrat, MIK-1282-500-1) in DEPC H2O for 5 min at RT. Following, the slides were then washed twice in 1x DEPC-PBS before dehydrating with ethanol series in 70% and 100% ethanol for 2 min each respectively. The slides were subsequently air dried at RT for 5 min before applying a Secure Seal chamber (Grace Bio-Labs) to each section. The sections were then rehydrated with 1x DEPC-PBS-T (KI Substrat, MIK1437-1L). To perform sample preparation the Cartana Neurokit was used (Cartana, Sweden). For reverse transcription 43.75 ul of Reaction Mix 1 (RM1), 1.25 ul of Enzyme 1 and 5.00ul of Enzyme 2 were mixed together and added to each tissue section in a secure seal chamber mounted on top of the tissue slides. Samples were incubated overnight at 37C. RM1 was then removed from the secure seal chambers and a post fixation solution containing 3.7% PFA in 1x DEPC-PBS was added to the samples and incubated at room temperature for 30 min. After post fixation, the samples were washed twice in 1x DEPC-PBS-T. For probe ligation, 36.0 ul of Reaction Mix 2 (RM2), 4.0 ul of Enzyme 3, 5.0 ul of Enzyme 4 and 100 nM of the Padlock probe (SEQ ID 1) were mixed and added into each secure seal chambers and incubated at 37C for 30 min followed by at 45C for 60 min. RM2 was then removed and samples were washed twice with 1x DEPC-PBS-T, after which the samples were ready for the in situ sequencing by hybridization reaction.
In Situ Sequencing by Hybridization of RCPs Immobilized on Glass Slides or in Tissue sections
[0235] In order to interrogate the first barcode subunit of the probe 100 .Math.l of a SBH mix containing 2x SSC, 20% or 30% Formamide and SBH- oligonucleotide G (CACA TGCGTCTATGTAGTGGAGCC TT AGAGAGTAGTACTTCCGACT, SEQ ID NO: 3), SBH-oligonucleotide A (CACA TGCGTCTATATAGTGGAGCC TT GTA GTA CAG CAG CAG CAT TGA GG, SEQ ID NO: 4), SBH- oligonucleotide T (CACA TGCGTCTATTTAGTGGAGCC TT CAA TCT AGT ATC AGT GGC GCA, SEQ ID NO: 5), SBH- oligonucleotide C (CACA TGCGTCTATCTAGTGGAGCC TT GGG CCT TAT TCC GGT GCT AT, SEQ ID NO: 6) and SBH-detection oligonucleotides Cy3 - AGTCGGAAGTACTACTCTCT (SEQ ID NO: 7), Cy5 - CCTCAATGCTGCTGCTGTACTAC (SEQ ID NO: 8), AF488 -TGCGCCACTGATACTAGATTG (SEQ ID NO: 9), and TexR -ATAGCACCGGAATAAGGCCC (SEQ ID NO: 10) was used. The SBH-oligonucleotides represent detection probes according to the present disclosure and invention. The detection oligonucleotides represent reporter probes as defined herein. The sequencing reaction was incubated for 30 min at 25° C., 37° C. or 45° C. The sequencing mix was then removed and the tissue sections were washed in PBS-T 0.05% twice. Subsequently, the tissue sections were mounted with mounting medium and a cover slip and imaged using 20x objective Zeiss microscope (Axio Z2).
Results
[0236] The first barcode subunit comprised A at the specific position that was being interrogated, and thus it was expected that the detection probe comprising T at the corresponding position would hybridise to the barcode subunit. The results are shown in
Example 2 - Linear Strand Displacement (LSD)
[0237] The LSD method represents the decoding of a continuous barcode sequence having sequential overlapping barcode positions, as described herein. The design of the LSD methods allows sequential toehold exchanges to be carried out in order to displace detection probes, thereby generating a decoding scheme that results in the identification of target nucleic acid molecules (e.g. genes in situ) without the need for stripping of detection probes (L-probes as used herein). In the main design, a detection probe in the form of an L-probe can hybridize to the target nucleic acid molecule, herein exemplified by an RCP, and a detection oligonucleotide (DO) (i.e. a reporter probe) is then allowed to hybridize and is subjected to imaging. The first detection L-probe consists of a reporter probe binding site, a unique toehold region (capable of hybridising to one barcode subunit of its target barcode position) and a common binding region (capable of hybridising to a second barcode subunit of the barcode position). The second detection L-probe that is designed to compete with the first detection L-probe also consists of a reporter probe binding site, a unique toehold region and a common region which it shares with the first L-probe (which hybridises to the overlapping barcode subunit, which is shared by the first and second barcode positions). The second L-probe will bind to the RCP via its unique toehold and compete with and displace the common binding site of the first L-probe. This results in the second L-probe fully occupying its RCP binding region and the first L-probe is left hybridized with just its unique toehold region. In order to complete the reaction, the unique toehold region of the first L-probe then spontaneously dissociates leaving the second L-probe (and its signal) as the only probe on the RCP. The unique toehold region of the second probe then becomes the common region it shares with the third L-probe and this will be used to set up another toehold exchange. This cycle can be continued for as many rounds as is necessary to decode the barcode.
[0238] The spontaneous displacement of the first probe during the toehold exchange can be driven forwards by the stringency of the hybridization buffer. The stringency of the hybridization buffer during the toehold exchange and the subsequent wash buffer are designed so that a 20 bp hybridization (i.e. the fully bound second detection probe) is less likely to dissociate compared to a 10bp hybridization (i.e. the partially bound first detection probe). In addition, the local concentration of the first probes is reduced to a level which is much lower than the concentration of the second probe, making it therefore much less likely to reverse the toehold exchange.
Methods
Mouse Tissue Section Preparation
[0239] Mouse strain C57BL/6 at 30 days age (P30) was euthanized and the olfactory bulb was dissected via cryosectioning. Cryosectioning was performed on ThermoFisher cryostat, at 10 .Math.m thickness. Sections were then adhered onto ThermoFisher Superfrost glass slides and stored at -70C until processing.
RCA Generation in Situ
Fixation and Permeabilization
[0240] The tissue slide was removed from -70° C. storage and allowed to thaw for 5 min at room temperature (RT). Fixation was then performed by incubating the slides in 3.7% PFA in 1X DEPC-PBS at RT for 5 min. The slide was then washed in 1X DEPC-PBS for 1 min at RT. This ensures that the PFA is completely removed before moving to the permeabilization step. The tissue sections were then permeabilized using 0.1 M HCl in DEPC-H2O for 1 min at RT and subsequently quickly washed twice in 1X DEPC-PBS. Following this, the slides were then dehydrated with an ethanol series in 70% and 100% ethanol for 2 min respectively before the slides are air-dried for 5 min at RT. A Secure Seal Chamber (Grace Bio Labs) are applied to each section and the sections are rehydrated with 1X DEPC-PBS-T before continuing with the reverse transcription step.
Reverse Transcription
[0241] Using CARTANA’s Neurokit, 43.75 .Math.l Reaction Mix (RM1), 1.25 .Math.l of Enzyme 1 (RNase Inhibitor) and 5.00 .Math.l of Enzyme 2 (Reverse Transcriptase) was added to each secure seal chamber and the samples were incubated in a humidity chamber at 37° C. overnight.
Probe Ligation
[0242] The reverse transcription was removed from the secure seal chambers and the slides were subjected to a post-fixation step using 3.7% PFA in DEPC-PBS for 30 min at RT. After the post-fixation step, the sections were quickly washed twice with DEPC-PBS-T. Using CARTANA’s Neurokit, 36.0 .Math.l Reaction Mix 2 (RM2), 4.0 .Math.l of Enzyme 3 (RNase H), 5.0 .Math.l of Enzyme 4 (Tth Ligase) and 100 nM of each padlock probe were added into each secure seal chamber and incubated at 37° C. for 30 min followed by a second incubation at 45° C. for 60 min. The ligation reaction mix was then removed from the secure seal chambers and the chambers were then washed twice with DEPC-PBS-T.
Rolling Circle Amplification
[0243] Using CARTANA’s Neurokit, 43.0 .Math.l of Reaction Mix 3 (RM3) and 5 .Math.l of Enzyme 5 (ϕ29 Polymerase) was added to the secure seal chambers and incubated at either 37° C. for 3 hrs or at 30° C. overnight. This is followed by the removal of the amplification reaction mix washed twice with DEPC-PBS-T. The secure seal chambers were then removed and the slides were then dehydrated with an ethanol series in 70% and 100% ethanol for 2 min respectively before the slides were air-dried for 5 min at RT. The sections were then used for in situ sequencing using the LSD design.
In Situ Sequencing of RCPs in Tissue Sections Using the LSD Design
Probe Design
[0244] The L-probes designed to hybridize to the RCP and displace each other consist of 3 distinct parts. The first part is the arm where the detection probe binds. This is 14-20bp long and encodes for a unique binding site specific to one detection probe, and hence colour. The second is a unique toehold region (7-10 bp) that allows the initial hybridization and a third part is a common binding sequence (7-10 bp) that is shared by the displacer probe (i.e. the subsequent detection L-probe).
Probe Hybridization
[0245] The sections are rehydrated with 2X SSC and the first probe mix was added at 100 nM in basic hybridization buffer (2.5X SSC + 5-20% Formamide (depending on hybridization length)) and incubated at 1 hr at RT. The sections are the washed twice with basic washing buffer (2X SSC in DEPC-H2O).
Detection Oligonucleotide (reporter Probe) Hybridization
[0246] After the L-probe hybridization, 100 nM detection oligo mix was added in basic hybridization buffer and allowed to hybridize for 30 min at RT. The sections were then washed twice with basic washing buffer before dehydrating the sections with an ethanol series in 70% and 100% ethanol for 2 min respectively, before the slides were air-dried for 5 min at RT. 10 .Math.l SlowFade Gold antifade reagent (Invitrogen) was then added to each section and covered with a coverslip. The slide was subjected to microscope imaging.
Toehold Exchange
[0247] After imaging, the coverslip was removed, and the slide was subjected to an ethanol series in 70% and 100% ethanol for 2 min respectively before air-drying the slide for 5 min at RT to remove the mounting media. The sections were then rehydrated with 2X SSC in DEPC-H2O. The second L-probe mix was then added at 200 nM in displacement hybridization buffer (2.5X SSC, 0.05% Tween-20 and 5-20% formamide conc. (depending on the hybridization length)). The sections were then incubated for 1 hr at RT and were subsequently washed with displacement wash buffer (1X DEPC-PBS-T and 10% formamide) twice for 10 min. After the toehold exchange, the sections were then subjected to detection oligo mix hybridization as described above.
Results
[0248] The proof-of-concept experiment was performed using a 20 bp hybridization length i.e. L-probes of 20 nucleotides in length, with a 10bp unique toehold region and a 10 bp common region.
[0249] As outlined in the description above, the “LSD Design” relies on each hybridization domain of the L-probe to have a unique toehold region and one sequence in common with the probe that is next in the toehold exchange sequence. Due to the design (
[0250] Compared to the discontinuous barcode sequence design, the LSD design utilizes a 7-10 bp unique toehold region and a 7-10bp common region. This hybridization length has a high specificity so it is very unlikely that non-specific hybridization and toehold exchange will occur.
[0251] Due to these factors, the LSD design is efficient, specific, cheap and compatible with downstream processes as it does not damage the tissue morphology due to high formamide usage.
Example 3 - In Situ Target Nucleic Acid Molecule Detection Using RCA and L-shaped detection probes
Methods
Mouse Tissue Section Preparation
[0252] Mouse strain C57BL/6 at 30 days age (P30) was euthanized and the olfactory bulb was dissected via cryosectioning. Cryosectioning was performed on ThermoFisher cryostat, at 10 .Math.m thickness. Sections were then adhered onto ThermoFisher Superfrost glass slides and stored at -70C until processing.
RCA Generation in Situ
Fixation and Permeabilization
[0253] The tissue slide was removed from -70° C. storage and allowed to thaw for 5 min at room temperature (RT). Fixation was then performed by incubating the slides in 3.7% PFA in 1X DEPC-PBS at RT for 5 min. The slide was then washed in 1X DEPC-PBS for 1 min at RT. This ensures that the PFA is completely removed before moving to the permeabilization step. The tissue sections were then permeabilized using 0.1 M HCl in DEPC-H.sub.2O for 1 min at RT and subsequently quickly washed twice in 1X DEPC-PBS. Following this, the slides were then dehydrated with an ethanol series in 70% and 100% ethanol for 2 min respectively before the slides are air-dried for 5 min at RT. A Secure Seal Chamber (Grace Bio Labs) are applied to each section and the sections are rehydrated with 1X DEPC-PBS-T before continuing with the reverse transcription step.
Reverse Transcription
[0254] Using CARTANA’s Neurokit, 43.75 .Math.l Reaction Mix (RM1), 1.25 .Math.l of Enzyme 1 (RNase Inhibitor) and 5.00 .Math.l of Enzyme 2 (Reverse Transcriptase) was added to each secure seal chamber and the samples were incubated in a humidity chamber at 37° C. overnight.
Probe Ligation
[0255] The reverse transcription was removed from the secure seal chambers and the slides were subjected to a post-fixation step using 3.7% PFA in DEPC-PBS for 30 min at RT. After the post-fixation step, the sections were quickly washed twice with DEPC-PBS-T. Using CARTANA’s Neurokit, 36.0 .Math.l Reaction Mix 2 (RM2), 4.0 .Math.l of Enzyme 3 (RNase H), 5.0 .Math.l of Enzyme 4 (Tth Ligase) and 100 nM of each padlock probe were added into each secure seal chamber and incubated at 37° C. for 30 min followed by a second incubation at 45° C. for 60 min. The ligation reaction mix was then removed from the secure seal chambers and the chambers were then washed twice with DEPC-PBS-T.
Rolling Circle Amplification
[0256] Using CARTANA’s Neurokit, 43.0 .Math.l of Reaction Mix 3 (RM3) and 5 .Math.l of Enzyme 5 (ϕ29 Polymerase) is added to the secure seal chambers and incubated at either 37° C. for 3 hrs or at 30° C. overnight. This is followed by the removal of the amplification reaction mix washed twice with DEPC-PBS-T. The secure seal chambers are then removed and the slides were then dehydrated with an ethanol series in 70% and 100% ethanol for 2 min respectively before the slides are air-dried for 5 min at RT. The sections can then be used for in situ sequencing using the LSD design.
In Situ Sequencing of RCPs in Tissue Sections Using the L-probe Design
Probe Design
[0257] The L-shaped detection probes (L-probes) designed to hybridize to the RCP probe binding site consists of 2 distinct parts. The first part is a 14-20nt long arm that recognizes the RCP. Each RCP is unique to a particular gene and hence this binding arm is specific for a specific gene. The second arm encodes for a reporter probe binding site. This sequence can differ depending on which reporter probe will bind. This arm consists of a 2nt linker and an 18nt reporter probe binding site.
Detection Probe Hybridization
[0258] The sections were rehydrated with 2X SSC and the first detection probe mix was added at 100 nM in basic hybridization buffer (2.5X SSC + 20% Formamide) and incubated at 1 hr at 20-37° C. The sections are the washed twice with basic washing buffer (2X SSC in DEPC-H.sub.2O).
Reporter Probe Hybridization
[0259] After the L-probe hybridization, 100 nM reporter probe mix was added in basic hybridization buffer and allowed to hybridize for 30 min at 20-37° C. The sections were then washed twice with basic washing buffer before dehydrating the sections with an ethanol series in 70% and 100% ethanol for 2 min respectively before the slides are air-dried for 5 min at RT. 10 .Math.l SlowFade Gold antifade reagent (Invitrogen) is then added to each section and covered with a coverslip. The slide was subjected to microscope imaging. After imaging, the L-probes and the reporter probes are removed and a new sequencing cycle can commence.
Results
[0260] The proof of concept experiments were carried out using L-probes with 20bp hybridization length, with a 20 bp RCP binding region.
[0261] The design of the L-probe hybridization method provides a general sequencing reaction with a decoding scheme that is both straightforward and highly flexible and results in the identification of genes of interest in situ. In the main design, an L-shaped detection probe can hybridize to the RCP, and a reporter probe is then allowed to hybridize and is subjected to imaging. The L-probes consists of a reporter probe binding site (20nt), a 2nt linker region and a RCP binding region (18nt) (
[0262] This design gives greater flexibility to change the decoding mechanism to include additional genes to the pool or reduce the number of genes in the pool. From
[0263] Having the decoding mechanism based on the L-probe pool and not the RCP means only a 20nt recognition site is required on the RCP and not multiple binding sites which are specific to a specific reporter probe. This keeps the padlock probe relatively small and easier to work with. The added benefit to having the decoding mechanism being based on the L-probe pool is that we can decide the length of the decoding barcode. This can range from 1 to 6 or more cycles without any need of increasing the padlock probe size and without the need for additional reporter probes to be designed. This also means that in order to encode for 4 reporter probes, 4 L-probes per gene will need to be designed. If an additional reporter probe will be used in the decoding scheme, another L-probe will have to be designed per gene to encode for this reporter probe. This, however, does provide us with an extra level of flexibility to implement an extra “base” if many genes will need to be sequenced.
Example 4 - Linear Strand Displacement With “back and Forth” Arrangement and Additional displacer probes (2-LSD)
[0264] This is a second iteration of the LSD design that is discussed above. In the original LSD method, a subsequent detection probe in the form of an L-probe was used to displace the previous detection probe, without the need for chemical stripping. In the 2-LSD design, the barcode comprises 2 binding sites, that is 2 barcode positions (i.e. recognition sites for 2 detection probes), which are arranged so as to facilitate a “back and forth” decoding approach. In this arrangement, only 2 binding sites are required on the target nucleic acid molecule (here an RCP) in order to fully decode the barcode. The binding sites on the RCP comprise a 5 bp common region (these are different to each other, but common among all RCPs). Both binding sites also contain a 5 bp region that is unique both to each binding site and each RCP. Lastly, there is a 10 bp region that is common to both binding sites but unique for each RCP (
[0265] The detection probes used in the 2-LSD method are modified L-probes which have been designed to have 2 overhangs, that is they are U-probes as defined above. They are slightly different from the detection probes used in the original LSD method in two ways. The first is the addition of a 10 bp displacer toehold overhang that acts as a toehold region for a short displacer probe to bind. This displacer toehold overhang is common for all detection probes which bind to a specific binding site (barcode position with common region). The presence of this additional displacer toehold region on the detection probes allows for displacement to occur simultaneously on both sides of the detection probe; at the displacer toehold site, the detection probe can be displaced by a displacer probe, and at the other end of the binding site, the detection probe can be displaced by the subsequent detection probe (as in the original LSD method).
[0266] The second change is that the detection probes are designed to bind specifically either to “binding site 1” or “binding site 2” (
[0267] As a typical sequencing workflow consist of 6 rounds of decoding, ie. 6 unique detection probes, the detection probes will be designed such that detection probes for cycles 1, 3 and 5 will bind binding site 1 and detection probes for cycles 2, 4 and 6 will bind to binding site 2. This means that a detection probe designed for cycle 2 will not be able to be used for cycle 5, for example.
[0268] The 2-LSD design removes the need to use chemical stripping to remove the detection probes. This is more gentle on the tissue samples which are being investigated, and allows the morphology of the sample to be preserved. The use of a “back-and-forth” design means that very similar binding sites to those used in the original LSD method can be used, thus retaining the size of the padlock probe (which is used to generate the RCP, and thus which contains a complementary copy of the barcode which appears in the RCP; in effect the barcode is coded into the padlock probe, and is copied into the RCP). In addition, the use of a common displacer toehold overhang region means that only 2 distinct displacer probes are required to displace all of the detection probes in the entire detection probe pool for all of the sequencing cycles. This reduces the costs of reagents, and makes the reaction more efficient.
Methods
Mouse Tissue Section Preparation
[0269] Mouse strain C57BL/6 at 30 days age (P30) was euthanized and the olfactory bulb was dissected via cryosectioning. Cryosectioning was performed on ThermoFisher cryostat, at 10 .Math.m thickness. Sections were then adhered onto ThermoFisher Superfrost glass slides and stored at -70° C. until processing.
RCA Generation in Situ
Fixation and Permeabilization
[0270] The tissue slide was removed from -70° C. storage and allowed to thaw for 5 min at room temperature (RT). Fixation was then performed by incubating the slides in 3.7% PFA in 1X DEPC-PBS at RT for 5 min. The slide was then washed in 1X DEPC-PBS for 1 min at RT. This ensured that the PFA was completely removed before moving to the permeabilization step. The tissue sections were then permeabilized using 0.1 M HCl in DEPC-H2O for 1 min at RT and subsequently quickly washed twice in 1X DEPC-PBS. Following this, the slides were then dehydrated with an ethanol series in 70% and 100% ethanol for 2 min respectively before the slides are air-dried for 5 min at RT. A Secure Seal Chamber (Grace Bio Labs) are applied to each section and the sections were rehydrated with 1X DEPC-PBS-T before continuing with the reverse transcription step.
Reverse Transcription
[0271] Using CARTANA’s Neurokit, 43.75 .Math.l Reaction Mix (RM1), 1.25 .Math.l of Enzyme 1 (RNase Inhibitor) and 5.00 .Math.l of Enzyme 2 (Reverse Transcriptase) was added to each secure seal chamber and the samples were incubated in a humidity chamber at 37° C. overnight.
Probe Ligation
[0272] The reverse transcription was removed from the secure seal chambers and the slides were subjected to a post-fixation step using 3.7% PFA in DEPC-PBS for 30 min at RT. After the post-fixation step, the sections were quickly washed twice with DEPC-PBS-T. Using CARTANA’s Neurokit, 36.0 .Math.l Reaction Mix 2 (RM2), 4.0 .Math.l of Enzyme 3 (RNase H), 5.0 .Math.l of Enzyme 4 (Tth Ligase) and 100 nM of each padlock probe were added into each secure seal chamber and incubated at 37° C. for 30 min followed by a second incubation at 45° C. for 60 min. The ligation reaction mix was then removed from the secure seal chambers and the chambers were then washed twice with DEPC-PBS-T.
Rolling Circle Amplification
[0273] Using CARTANA’s Neurokit, 43.0 .Math.l of Reaction Mix 3 (RM3) and 5 .Math.l of Enzyme 5 (ϕ29 Polymerase) was added to the secure seal chambers and incubated at either 37° C. for 3 hrs or at 30° C. overnight. This was followed by the removal of the amplification reaction mix, and a step of washing twice with DEPC-PBS-T. The secure seal chambers were then removed and the slides were then dehydrated with an ethanol series in 70% and 100% ethanol for 2 min respectively before the slides were air-dried for 5 min at RT. The sections were then able to be used for in situ sequencing using the LSD design.
In Situ Sequencing of RCPs in Tissue Sections Using the 2-LSD Design
Probe Design
[0274] The detection probes were designed to hybridize either to binding site 1 or binding site 2 on the RCP. The 2 designs were identical except for the orientation. The detection probe comprised a 10 bp displacer toehold overhang region which, combined with a 5 bp sequence in the RCP recognition domain, functioned as a generic toehold region common to all detection probes. The RCP binding region, comprising the aforementioned 5 bp toehold plus an additional 15 bp, spanned 20 bp and the reporter probe binding site was 20 bp in length. The detection probe had a GC content of 50%. For binding site 1, the displacer toehold overhang region was on the left and the reporter probe binding region was on the right of the RCP binding domain. For the detection probe binding to binding site 2, the reporter probe binding region was on the left side of the RCP binding domain and the displacer toehold overhang region was on the right.
[0275] The barcode design of the padlock probe comprised a 30 bp detection probe binding domain. It had 2 common regions at either end of the barcode, each being 5 bp in length. These common regions were different to each other but were common for all padlock probes, regardless of their targets. There were 2 unique regions that were 5 bp in length and were located next to the common regions. These unique regions were unique to each binding site. In the middle of the 30 bp detection probe binding region, there was a 10 bp binding region that was shared between the two binding sites. This ensured the ability for one detection probe to partially displace another detection probe. Each binding site consisted of 1 common region (5 bp), 1 unique region (5 bp) and the 10 bp common region, thus providing a 20 bp binding region for the detection probe to hybridize to.
Probe Hybridization
[0276] The sections were rehydrated with 1X PBS and the first probe mix was added at 100 nM in basic hybridization buffer (2.5X SSC + 5-20% Formamide (depending on hybridization length)) and incubated at 1 hr at 37C. The sections were then washed twice with basic washing buffer (1X PBS).
Detection Oligo Hybridization
[0277] After the detection probe hybridization, 100 nM detection oligo mix was added in basic hybridization buffer and allowed to hybridize for 30 min at 37° C. The sections were then washed twice with basic washing buffer before dehydrating the sections with an ethanol series in 70% and 100% ethanol for 2 min respectively before the slides were air-dried for 5 min at RT. 10 .Math.l SlowFade Gold antifade reagent (Invitrogen) was then added to each section and covered with a coverslip. The slide was subjected to microscope imaging.
Displacement of Detection Probes Using the 2-LSD Approach
[0278] After imaging, the coverslip was removed, and the slide was subjected to an ethanol series in 70% and 100% ethanol for 2 min respectively before air-drying the slide for 5 min at RT to remove the mounting media. The sections were then rehydrated with 1X PBS. The second detection probe and a displacer strand mix was then added, both at 200 nM final concentration in displacement hybridization buffer ( 2.5X SSC, 0.05% Tween-20 and 5-20% Formamide conc. (depending on the hybridization length)). The sections were then incubated for 1 hr at 37° C. and were subsequently washed with displacement wash buffer (1X DEPC-PBS-T and 20% formamide) twice for 10 min at room temperature. After the displacement reaction was complete, the sections were then subjected to DO mix hybridization as described above.
Results
Displacement of Detection Probes Under Different Buffer Conditions
[0279] The buffer used to displace one detection probe with the next in the sequencing cycle is key to an efficient and complete displacement during the 2-LSD method. Variations of a standard hybridization buffer were used in order to test which components are necessary to efficiently displace the detection probe. Three cycles of sequencing involving 2 displacement steps were performed in order to observe a color change as well as the recovery of the signal from the first cycle in the third cycle. All the buffer compositions tested were deemed successful at displacing the detection probes in cycle 2 and cycle 3 and lead to a switch from Cy5 to Cy3 and back to Cy5. From the results, it was concluded that the displacement in the second cycle (from Cy5 to Cy3) is less efficient for condition 3 (2.5X SSC + 20% Formamide). This can be seen by analyzing the child:parent ratio (
[0280] The recovery of the signal in the Cy5 channel (i.e. the third cycle) was lower in terms of the signal intensity (
2-LSD Cycling of 2 Displacement Cycles Using “back-and-forth” Method
[0281] Utilizing the optimal displacement buffer composition as outlined above, a 3 cycle 2-LSD set-up was used to investigate how efficiently the detection probes are displaced.