US 2025/0154564 A1Published application

Methods for performing spatial profiling of biological molecules

AssigneeCentrillion Technology Holdings Corporation

InventorsWei Zhou, Suzy Chang, T. Scott Pollom, Jeremy Edwards, Justin Costa, Yaning Li, Xun Ding

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Abstract

The present disclosure provides methods, devices and systems that enable determination of spatial information of biological molecules by reacting the biological molecules with a zipcode array. In some examples, the zipcode array may code for the spatial positions of biological molecules attached to distinct positions on the zipcode array. In some examples, the spatial positions are 2-dimensional. In some cases, the spatial positions are 3-dimensional. In some examples, the present disclosure provides methods to detect spatial gene expression. In some examples, the present disclosure provides methods to detect spatial distribution of proteins.

Claims32

Dependent claims

A method for detecting spatial distribution of biomolecule expression within a biological sample, comprising:

(a) providing a substrate having a plurality of distinct locations, wherein each distinct location of the plurality of distinct locations comprises a first coordinate and a second coordinate;
(b) attaching a plurality of oligonucleotide zipcodes to each distinct location, thereby encoding the first coordinate and the second coordinate by the plurality of oligonucleotide zipcodes, wherein each zipcode of the plurality of oligonucleotide zipcodes encodes the first coordinate and the second coordinate, and comprises:
(i) a bottom adapter attached to the distinct location;
(ii) a lower zipcode attached to the bottom adapter;
(iii) a separator sequence attached to the lower zipcode;
(iv) an upper zipcode attached to the separator sequence;
and
(v) a top adapter attached to the upper zipcode;
wherein the separator sequence comprises a sequence selected from the group consisting of GGG, CCC and TT,
wherein different zipcodes attached to different distinct locations have a long-range minimum edit distance of 5, and
wherein a long-range minimum edit distance of 5 means that if two sequences are >5 locations apart on the substrate, there must be >5 changes needed to convert one sequence into another sequence;
(c) contacting a biological sample comprising a plurality of biomolecules with the plurality of zipcodes, thereby attaching at least a fraction of the plurality of zipcodes with at least a fraction of the plurality of biomolecules and generating a plurality of tagged biomolecules;
(d) sequencing the plurality of tagged biomolecules and determining the first coordinate and the second coordinate for at least the fraction of the plurality of tagged biomolecules, thereby determining the spatial distribution of the plurality of biomolecules within the biological sample.

10 more elements

A zipcode array, comprising:

(a) a first location comprising a first coordinate and a second coordinate;
(b) a second location comprising a third coordinate and a fourth coordinate;
(c) a plurality of first oligonucleotide zipcodes attached to the first location, thereby encoding the first coordinate and the second coordinate by the plurality of first oligonucleotide zipcodes, wherein each first zipcode of the plurality of first oligonucleotide zipcodes encodes the first coordinate and the second coordinate, and comprises:
(i) a first bottom adapter attached to the first location;
(ii) a first lower zipcode attached to the first bottom adapter;
(iii) a first separator sequence attached to the first lower zipcode;
(iv) a first upper zipcode attached to the first separator sequence;
and
(v) a first top adapter attached to the first upper zipcode;
and
(d) a plurality of second oligonucleotide zipcodes attached to the second location, thereby encoding the third coordinate and the fourth coordinate by the plurality of second oligonucleotide zipcodes, wherein each second zipcode of the plurality of second oligonucleotide zipcodes encodes the third coordinate and the fourth coordinate, and comprises:
(i) a second bottom adapter attached to the first location;
(ii) a second lower zipcode attached to the second bottom adapter;
(iii) a second separator sequence attached to the second lower zipcode;
(iv) a second upper zipcode attached to the second separator sequence;
and
(v) a second top adapter attached to the second upper zipcode,
wherein each of the first separator sequence and the second separator sequence comprise a sequence selected from the group consisting of GGG, CCC and TT,
wherein different zipcodes attached to different distinct locations have a long-range minimum edit distance of 5, and
wherein a long-range minimum edit distance of 5 means that if two sequences are >5 locations apart on the substrate, there must be >5 changes needed to convert one sequence into another sequence.

17 more elements

A method for detecting spatial distribution of a plurality of ribonucleic acid molecules in a biological sample, comprising:

(a) contacting a first surface comprising a plurality of first oligonucleotides with a biological sample comprising a plurality of ribonucleic acid molecules;
(b) extending a fraction of the plurality of first oligonucleotides by a transcriptase using the plurality of ribonucleic acid molecules as templates, thereby generating a plurality of second oligonucleotides, each of the plurality of second oligonucleotides comprising a fragment of complementary DNA (cDNA) of one of the plurality of ribonucleic acid molecules;
(c) contacting a zipcode array comprising a plurality of zipcode oligonucleotides with the plurality of second oligonucleotides in the presence of a polymerase, thereby extending the plurality of second oligonucleotides and generating a plurality of third oligonucleotides, each of the plurality of third oligonucleotides comprising one of the plurality of second oligonucleotides and a complementary sequence of one of the plurality of zipcode oligonucleotides,
wherein the zipcode array comprises:
(i) a first location comprising a first coordinate and a second coordinate;
(ii) a second location comprising a third coordinate and a fourth coordinate;
and
(iii) a plurality of first oligonucleotide zipcodes attached to the first location thereby encoding the first coordinate and the second coordinate by the plurality of first oligonucleotide zipcodes, wherein each first zipcode of the plurality of first oligonucleotide zipcodes encodes the first coordinate and the second coordinate, and comprises:
(A) a first bottom adapter attached to the first location;
(B) a first lower zipcode attached to the first bottom adapter;
(C) a first separator sequence attached to the first lower zipcode;
(D) a first upper zipcode attached to the first separator sequence;
and
(E) a first top adapter attached to the first upper zipcode;
and
(iv) a plurality of second oligonucleotide zipcodes attached to the second location, thereby encoding the third coordinate and the fourth coordinate by the plurality of second oligonucleotide zipcodes, wherein each second zipcode of the plurality of second oligonucleotide zipcodes encodes the third coordinate and the fourth coordinate, and comprises:
(A) a second bottom adapter attached to the first location;
(B) a second lower zipcode attached to the second bottom adapter;
(C) a second separator sequence attached to the second lower zipcode;
(D) a second upper zipcode attached to the second separator sequence;
and
(E) a second top adapter attached to the second upper zipcode,
wherein each of the first separator sequence and the second separator sequence comprise a sequence selected from the group consisting of GGG, CCC and TT,
wherein different zipcodes attached to different distinct locations have a long-range minimum edit distance of 5, and
wherein a long-range minimum edit distance of 5 means that if two sequences are >5 locations apart on the substrate, there must be >5 changes needed to convert one sequence into another sequence;
(d) separating the first surface comprising the plurality of third oligonucleotides from the zipcode array;
and
(e) sequencing the plurality of third oligonucleotides, thereby determining the spatial distribution of the plurality of ribonucleic acid molecules within the biological sample.

25 more elements

Dependencies are inferred from the claim text.

Facts

Application: 18/668,000Filed 17 May 2024
Priority: 23 May 2017
Published: 15 May 2025
Term ends: Not estimatedThe source records don’t support an estimate.

Classification11 codes

C12Q 1/6837
Enzymatic or biochemical coupling of nucleic acids to a solid phase: using probe arrays or probe chips
C12N 15/10
Recombinant DNA-technology: processes for the isolation, preparation or purification of DNA or RNA
C12Q 1/6841
Hybridisation assays: in situ hybridisation
C12Q 1/6809
Involving nucleic acids: methods for determination or identification of nucleic acids involving differential detection

Family

Application62/509,764Us provisional application · filed 23 May 2017
Application62/509,765Us provisional application · filed 23 May 2017
Application62/509,766Us provisional application · filed 23 May 2017
Application62/510,358Us provisional application · filed 24 May 2017
Application62/510,356Us provisional application · filed 24 May 2017
Application62/510,353Us provisional application · filed 24 May 2017
Application62/568,200Us provisional application · filed 4 Oct 2017
Application16/614,677Continuation · filed 18 Nov 2019
Application18/668,000Continuation
US 2020/0370105 A1Published application
US 2025/0154564 A1This patent

From the US filing history. Foreign family members aren’t listed.