阅读说明：本技术 系统 (System for controlling a power supply ) 是由 C·W·布朗三世 W·J·布莱克 R·K·丹达 于 2020-03-20 设计创作，主要内容包括：本公开描述了允许灵敏检测目的核酸(即,核苷酸序列是或包含靶序列的核酸)的技术。(The present disclosure describes techniques that allow for sensitive detection of a nucleic acid of interest (i.e., a nucleic acid whose nucleotide sequence is or comprises a target sequence).)

1. a system, the system comprising:

a plurality of nucleic acid molecules having different nucleotide sequences;

a set of adaptor oligonucleotides, the set of adaptor oligonucleotides comprising:

a first adaptor oligonucleotide having a nucleotide sequence comprising a template element and a first target-hybridizing element; and

a second adaptor oligonucleotide having a nucleotide sequence comprising a second target-hybridizing element and a Cas recognition element; and

optionally one or more bridging oligonucleotides, the nucleotide sequence of which is or includes one or more additional target-hybridizing elements,

wherein the target-hybridizing elements bind to different portions of a common target site such that when the plurality of nucleic acid molecules includes at least one nucleic acid molecule whose nucleotide sequence includes the target site, the set of linking oligonucleotides then hybridizes to the target site and forms a nicked nucleic acid strand that is readily ligatable with a ligase, thereby producing a linked strand.

2. The system of claim 1, further comprising a ligase.

3. The system of claim 1, wherein one or more of the oligonucleotides are associated with a solid support.

4. The system of claim 1, wherein the plurality of nucleic acids are associated with a solid support.

5. The system of claim 1, further comprising a second set of linking oligonucleotides, different from the first set of linking oligonucleotides, directed at a second target site.

6. The system of claim 5, wherein the template elements of the first and second set of adaptor oligonucleotides are the same.

7. The system of claim 5, wherein the template elements of the first and second set of adaptor oligonucleotides are different.

8. The system of any one of claims 5-7, wherein the Cas recognition elements of the first and second sets of adaptor oligonucleotides are the same.

9. The system of any one of claims 5-7, wherein the Cas recognition elements of the first and second sets of adaptor oligonucleotides are different.

10. The system of claim 1, wherein the template element is or comprises a promoter or a complement thereof.

11. The system of claim 1, wherein the template element is or comprises an origin of replication or a complement thereof.

12. The system of claim 1, wherein the template element is or comprises a binding site for a first extendable primer.

13. The system of claim 12, further comprising a first extendable primer.

14. The system of claim 12 or claim 13, further comprising a DNA polymerase.

15. The system of claim 12 or claim 13, wherein the second linking oligonucleotide has a sequence that includes the complement of the binding site of the second extendable primer.

16. The system of claim 15, further comprising a second extendable primer.

17. The system of claim 16, further comprising a DNA polymerase.

18. The system of claim 17, wherein the DNA polymerase is thermostable.

19. The system of any one of the preceding claims, further comprising an RNA polymerase.

20. A set of adaptor oligonucleotides, the set of adaptor oligonucleotides comprising:

a first adaptor oligonucleotide having a nucleotide sequence comprising a template element and a first target-hybridizing element; and

a second adaptor oligonucleotide having a nucleotide sequence comprising a second target-hybridizing element and a Cas recognition element; and

optionally one or more bridging oligonucleotides, the nucleotide sequence of which is or includes one or more additional target-hybridizing elements,

wherein the target-hybridizing elements bind to different portions of a common target site such that when the set of linking oligonucleotides is brought into contact with a nucleic acid molecule whose nucleotide sequence includes the target site, the set of linking oligonucleotides then hybridizes to the target site and forms a nicked nucleic acid strand that is readily ligatable with a ligase, thereby producing a ligated strand.

21. A method, comprising the steps of:

contacting the set of adaptor oligonucleotides of claim 20 with a nucleic acid sample under conditions that allow for simultaneous hybridization of the oligonucleotides in the set of adaptor oligonucleotides to one or more individual nucleic acid molecules in the nucleic acid sample;

simultaneously or subsequently contacting the set of adaptor oligonucleotides with a ligase such that a ligated strand is generated if the nucleic acid sample comprises at least one nucleic acid molecule whose nucleotide sequence includes a target site.

22. The method of claim 21, wherein the template element is or comprises a binding site for an extendable primer, and the method further comprises the steps of:

hybridizing the extendable primer to the connecting strand; and

extending the extendable primer such that a duplex is formed.

23. The method of claim 22, wherein the template element is further or comprises a promoter, and the method comprises the steps of:

(ii) transcribing from the promoter such that a transcript is produced that includes the Cas recognition element.

24. The method of claim 22, further comprising the steps of: replicating one or both strands of the duplex such that a population of single-stranded or double-stranded nucleic acid molecules are generated that each include the Cas recognition element.

25. The method of claim 23 or claim 24, further comprising the step of: contacting the Cas recognition element with:

a Cas enzyme characterized by collateral activity; and

a guide RNA that binds to the Cas recognition element,

the contacting is performed when a nucleic acid probe is susceptible to cleavage by the Cas enzyme side branch present; and

detecting cleavage of the nucleic acid probe.

26. The method of claim 25, wherein the detecting indicates that a target nucleic acid whose nucleotide sequence includes the target site is present in the nucleic acid sample, and the method further comprises:

quantifying the amount of target nucleic acid present in the nucleic acid sample.

Background

The development of techniques that can specifically detect target nucleic acids is becoming increasingly important, especially when present in very low abundance.

Disclosure of Invention

The present disclosure describes techniques that allow for sensitive detection of a nucleic acid of interest (i.e., a nucleic acid whose nucleotide sequence is or comprises a target sequence).

In some embodiments, provided techniques exploit the nucleic acid cleavage activity characteristics of certain Cas enzymes; in many embodiments, provided techniques exploit the collateral cleavage activity of certain Cas enzymes. The present disclosure identifies the source of problems associated with certain previously developed techniques that exploit the collateral cleavage activity of Cas. The present disclosure addresses such issues and, in addition, provides techniques with unexpected benefits and/or capabilities relative to alternative available systems.

Importantly, in many embodiments, the present disclosure provides techniques for decoupling sequence detection from cleavage activation by Cas enzymes. Thus, the present disclosure represents a significant departure from most Cas-based technology systems, focusing on the Cas enzyme's markers: their activity can be directed specifically to any target sequence by manipulation of the guide RNA sequence.

In some embodiments, the present disclosure provides insight that by taking the opposite approach-using a Cas system to detect a particular sequence but not engineering a Cas guide RNA to hybridize to that sequence, certain advantages can be achieved and/or problems can be solved. In some embodiments, the present disclosure teaches the use of a ligation-based system for initial target nucleic acid hybridization, wherein the components of the ligation-based system are designed such that the ligation event results in one or more nucleic acid molecules or templates that are activating nucleic acids for Cas collateral cleavage activity. The system provided allows the activated nucleic acid to have a sequence that is not required to be found in the original target nucleic acid. Thus, in some embodiments, the present disclosure provides techniques that can utilize one or a relatively small number of guide RNA sequences to detect a variety of different target nucleic acid targets (including targets that differ from other nucleic acid sequences by a single base).

Alternatively or additionally, in some embodiments, the present disclosure provides strategies that allow nucleic acid detection without amplification of the initial target nucleic acid, even with significant (e.g., attomolar) sensitivity.

Among other things, advantages of the provided techniques include (i) the possibility of eliminating pre-amplification of target nucleic acids, thereby removing potential sources of sequence mutations that may misinterpret assay results (e.g., by generating false positive and/or false negative readings); (ii) uncoupling target detection from activation of Cas collateral activity avoids the need to specifically design different grnas for each target sequence of interest; (iii) uncoupling activation of Cas side branch activity from target detection further allows flexibility in selecting Cas systems, as one or both of DNA and RNA that activate nucleic acids can be generated and/or utilized; (iv) ligation specificity enables single base discrimination; (v) decoupling the activation of target detection from Cas collateral activity allows, among other things, multimerization of Cas activation sequences (i.e., sequences bound by grnas/crrnas), thereby potentially allowing activation of multiple Cas enzymes from a single ligation product (or template or copy thereof); and (vi) combinations thereof.