US 2023/0416828 A1Published application

Rna and dna analysis using engineered surfaces

AssigneeAlida Biosciences, Inc.

InventorsGudrun STENGEL, Hua YU, Andrew PRICE, Jerome SANTOS, Yu-Hsien HWANG-FU, Byron PURSE

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Abstract

Provided herein are compositions and methods for the multiplexed profiling of RNA and DNA modifications across transcriptomes and genomes, respectively. The methods combine molecular recognition of non-canonical features (e.g., base modifications, backbone modifications, lesions, and/or structural elements) of a target nucleic acid with a step of writing the information from this recognition event into the neighboring genetic sequence of the target nucleic acid using a barcode. The resultant barcoded nucleic acids are then converted into sequencing libraries and read by DNA/RNA sequencing methods. This step reveals the sequence of the barcode, which is correlated with the non-canonical feature in the target nucleic acid(s). The high throughput profiling methods described herein allow for identification and/or localization of one or more modifications in a target nucleic acid. The methods also allow for identification of the nature and location of several or all DNA/RNA modifications in parallel.

Claims30

Dependent claims

A method for detecting a plurality of non-canonical features in a plurality of target nucleic acids, the method comprising:

(i) providing a plurality of target nucleic acids by reverse transcribing target RNA molecules to form DNA-RNA heteroduplex molecules or providing target double-stranded DNA molecules;
(ii) contacting a solution comprising the plurality of target nucleic acids with a composition comprising:
a substrate,
a binding domain coupled to the substrate via a first linker or a secondary recognition element,
mosaic end (ME) adapters coupled to the substrate via a second linker or secondary recognition element, and
a transposase,
wherein the transposase is loaded to the immobilized ME adapters,
wherein the binding domain binds specifically to a non-canonical feature of a DNA or an RNA,
wherein at least one of the ME adapters comprises a nucleic acid barcode sequence unique to the non-canonical feature;
or
the substrate,
the binding domain coupled to the substrate via a linker or secondary recognition element, and
the transposase coupled to the binding domain,
wherein the transposase is loaded with ME adapters,
wherein the binding domain binds specifically to a non-canonical feature of a DNA or an RNA,
wherein at least one of the ME adapters comprises a nucleic acid barcode sequence unique to the non-canonical feature;
(iii) transferring, using transposase, two adapters, at least one of them comprising the nucleic acid barcode, to a double-stranded target nucleic acid comprising the non-canonical feature to generate barcoded target nucleic acids,
(iv) amplifying the barcoded target nucleic acids;
and
(v) sequencing the barcoded target nucleic acids,
wherein steps (ii) and (iii) are performed concurrently or sequentially.

18 more elements

A method for detecting a plurality of non-canonical features in a plurality of target nucleic acids, the method comprising:

(i) contacting a solution comprising the plurality of target nucleic acids with one or more compositions,
wherein the one or more compositions are each independently selected from:
(a) a composition comprising:
a substrate,
a binding domain coupled to the substrate via a first linker or via a secondary recognition element, and
an adapter coupled to the secondary recognition element or to the substrate via a second linker,
wherein the binding domain is configured to bind specifically to a non-canonical feature of a DNA or an RNA;
wherein the adapter comprises a nucleic acid barcode sequence unique to the non-canonical feature;
and
(b) a composition comprising:
a substrate,
a binding domain coupled to the substrate via a first linker or via a plurality of secondary recognition elements, and
an adapter coupled to one or more of the plurality of secondary recognition elements or to the substrate via a second linker,
wherein the binding domain is configured to bind specifically to a non-canonical feature of a DNA or an RNA;
wherein the adapter comprises a nucleic acid barcode sequence unique to the non-canonical feature;
(ii) performing one of (a) and (b):
(a) transferring the nucleic acid barcode to a target nucleic acid comprising the non-canonical feature to generate a barcoded target nucleic acid,
(b) generating a barcoded copy of the target nucleic acid comprising the non-canonical feature;
(iii) amplifying the barcoded target nucleic acids;
and
(iv) sequencing the barcoded target nucleic acids,
wherein steps (i) and (ii) are performed sequentially or concurrently.

19 more elements

A method for detecting a plurality of non-canonical features in a plurality of target nucleic acids, the method comprising:

(i) contacting a solution comprising the plurality of target nucleic acids with a plurality of compositions;
wherein the plurality of compositions are each independently selected from:
(a) a composition comprising:
a substrate;
a binding domain coupled to the substrate via a first linker or via a secondary recognition element;
and
an adapter coupled to the secondary recognition element or to the substrate via a second linker,
wherein the binding domain is configured to bind specifically to a non-canonical feature of a DNA or an RNA;
wherein the adapter comprises a nucleic acid barcode sequence unique to the non-canonical feature;
(b) a composition comprising:
a substrate,
a secondary recognition element coupled to the substrate via a first linker, and
an adapter coupled to the secondary recognition element or to the substrate via a second linker, and
a binding domain,
wherein the binding domain is configured to bind specifically to a non-canonical feature of a DNA or an RNA, and wherein the binding domain is immobilized by the secondary recognition element, and
wherein the adapter comprises a nucleic acid barcode sequence unique to the non-canonical feature;
(c) a composition comprising:
a substrate,
a secondary recognition element,
a binding domain coupled to the substrate via a secondary recognition element,
an adapter coupled to the substrate via a linker,
wherein the binding domain is configured to bind specifically to a non-canonical feature of a DNA or an RNA, and
wherein the adapter comprises a nucleic acid barcode sequence unique to the non-canonical feature;
(d) a composition comprising:
a substrate,
a secondary recognition element coupled to the substrate,
a binding domain coupled to the substrate via a linker,
an adapter coupled to the substrate via the secondary recognition element,
wherein the binding domain is configured to bind specifically to a non-canonical feature of a DNA or an RNA, and
wherein the adapter comprises a nucleic acid barcode sequence unique to the non-canonical feature;
(e) a composition comprising:
a substrate,
a binding domain coupled to the substrate via a first linker or via a plurality of secondary recognition elements, and
an adapter coupled to one or more of the plurality of secondary recognition elements or to the substrate via a second linker,
wherein the binding domain is configured to bind specifically to a non-canonical feature of a DNA or an RNA;
wherein the adapter comprises a nucleic acid barcode sequence unique to the non-canonical feature;
wherein the number of the plurality of compositions contacted in step (i) is equal to or greater than the number of non-canonical features,
wherein the binding domains of the plurality of compositions each bind to different non-canonical features of a DNA or RNA or wherein multiple binding domains bind to the same non-canonical feature of a DNA or RNA;
and
wherein the adapters of the plurality of compositions each comprise a nucleic acid barcode sequence unique to the non-canonical feature bound specifically by the binding domain of that composition or unique to the binding domain;
(ii) performing one of the following:
(a) transferring the nucleic acid barcode sequences of each of the plurality of compositions to a plurality of target nucleic acids, or
(b) generating barcoded copies of the plurality of target nucleic acids;
(iii) amplifying the barcoded target nucleic acids;
and
(iv) sequencing the barcoded target nucleic acids.

43 more elements

Dependencies are inferred from the claim text.

Facts

Application: 18/343,028Filed 28 Jun 2023
Priority: 24 Nov 2021
Published: 28 Dec 2023
Term ends: Not estimatedThe source records don’t support an estimate.

Classification12 codes

C12Q 1/6804
Involving nucleic acids: nucleic acid analysis using immunogens
C12Q 1/6883
Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes: for diseases caused by alterations of genetic material
C12N 9/22
Acting on ester bonds: ribonucleases [RNase]; Deoxyribonucleases [DNase]
C12N 15/1065
Isolating an individual clone by screening libraries: preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis,…

Family

Application63/282,808Us provisional application · filed 24 Nov 2021
Application63/388,036Us provisional application · filed 11 Jul 2022
Application18/343,028Continuation
US 12,258,631 B2Related patent
US 2023/0416828 A1This patent

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