WO2017197147A2

WO2017197147A2 - Molecular beacon comprising prefabricated components and associated products, processes, and methods of use

Info

Publication number: WO2017197147A2
Application number: PCT/US2017/032210
Authority: WO
Inventors: Robert G. Atkinson
Original assignee: Atkinson Robert G
Priority date: 2016-05-11
Filing date: 2017-05-11
Publication date: 2017-11-16
Also published as: WO2017197147A3

Abstract

The present invention relates to improved dissociative molecular beacons constructed from a combination of prefabricated and customized components, and associated products, processes, and methods for their use. The present invention comprises a molecular beacon that is quickly and easily assembled from a kit of parts comprising three components: a pair of target-neutral labeled oligos, each with either a fluorophore or a quencher attached; and a non-labeled target-specific oligo. The labeled oligos of the present invention comprise specific structural ligation site regions and short loop regions. Ligation of the three components forms the molecular beacons of the present invention. In an embodiment, the present invention further comprises a non-labeled target-specific oligo including a toehold region adjacent to a linker region.

Description

MOLECULAR BEACON COMPRISING PREFABRICATED COMPONENTS AND ASSOCIATED PRODUCTS, PROCESSES, AND METHODS OF USE

RELATED APPLICATIONS

This application claims priority to U.S. provisional application serial number 62/334,933, filed on May 11, 2016, titled MOLECULAR BEACON

COMPRISING PREFABRICATED COMPONENTS AND ASSOCIATED PRODUCTS, PROCESSES, AND METHODS OF USE. The entire contents of the above-referenced application is incorporated by reference herein and made part of this specification. FIELD OF THE INVENTION

The present invention relates to improved dissociative molecular beacons constructed from a combination of prefabricated and customized components, and associated products, processes, and methods for their use. The present invention comprises a molecular beacon that is quickly and easily assembled from a kit of parts comprising three components: a pair of target-neutral labeled oligos, each with either a fluorophore or a quencher attached; and a non-labeled target-specific oligo. The labeled oligos of the present invention comprise specific structural ligation site regions and short loop regions. Ligation of the three components forms the molecular beacons of the present invention. In an embodiment, the present invention further comprises a non- labeled target-specific oligo including a toehold region adjacent to a linker region.

BACKGROUND

Molecular beacons are today a workhorse fluorescent probe technology for the detection and quantification of nucleic acids. See Tyagi, S. & Kramer, F. R. Molecular beacons: Probes that fluoresce upon hybridization, Nature biotechnology 14, 303-308, doi:DOI 10.1038/nbt0396-303 (1996) ("Tyagi"); Wang, K. et al. Molecular engineering of DNA: molecular beacons, Angewandte Chemie 48, 856-870, doi: 10.1002/anie.200800370 (2009) ("Wang K."); and Wikipedia, Molecular beacon, <https:/yenA¥ikipedia.org/wiki/Molecular beacon> (2016). Molecular beacons are currently used in a wide range of contexts, from genetic screening and monitoring of intra-cellular mRNA production to the quantitative monitoring of DNA amplification. See Wang K.; Yang, C. J. & Tan, W., Molecular Beacons, Springer- Verlag Berlin Heidelberg (2013). A molecular beacon is comprised of a hairpin DNA oligonucleotide consisting of a complementary stem region at either end bookending a central loop region, the latter containing a portion which is complementary to the intended nucleic acid target of the probe. See Wikipedia, Oligonucleotide,

<https:/yen.wikipedia.org/V/iki/Oligonucieotide> (2016); Wikipedia, Base pair,

- hs ip /. en '.vs ki cxiia or /u i kt Base __pai ; ' (2016). To the ends of the oligo (at the extremes of the stem regions) are affixed two molecular labels, one a fluorophore and the other a quencher. See Wikipedia, Fluorophore,

<b.ttps://½. wjkipedia.org/wi.ki/Fluorophore> (2016); Wikipedia, Dark quencher, <https://en. wikipedia. org/wiki/TJark _^quenchei^ (2016).

To a first approximation, a molecular beacon can be found in just one of two states: a quiescent state, where it is unbound to its intended target, or an active state, where the loop region has hybridized to its intended target strand (Figure 1). In the quiescent state, the two complementary stem regions hybridize to each other, and the beacon overall takes on a shape commonly said to resemble a hairpin. See

Wikipedia, Hairpin, <https://en.wikipeciia.org/wi ki /Hairpm> (2016). This brings the fluorophore and quencher adjacent to each other, which significantly diminishes the fluorescence of the fluorophore. In the active state, the binding of the loop region to its target is more thermodynamically favorable than the binding of the complementary stem regions since, among other considerations, the loop region contains a greater number of nucleotides than the stem regions, and the hairpin opens up. This separates the fluorophore and the quencher, which increases fluorescence relative to the quiescent state.

Despite the fact that molecular beacons are structurally very simple, and that the synthesis of DNA oligos is today highly automated and surprisingly cheap and efficient, the manufacture of molecular beacons remains both time-consuming and relatively expensive. This is almost entirely due to the need to chemically affix the fluorophore and quencher labels to the ends of the DNA oligo, as this requires both an attachment chemistry which is distinct from that which is used to construct the oligo itself, as well as additional purification steps to refine the result.

As an illustration, consider the original 25-nucleotide molecular beacon illustrated in Figure 2. See, e.g., Tyagi, above. Integrated DNA Technologies (IDT), a leading manufacturer of synthetic DNA and RNA, quotes $405 as the price to manufacture 250 nmol of this molecular beacon using their least expensive fluorophore and quencher (a green FAM fluorophore and an Iowa Black quencher, respectively). Moreover, the time required to complete the order is listed as five to seven business days. In contrast, 250 nmol of just the oligo itself is quoted as $25 and available with overnight delivery. The liberal use of molecular beacons can thus become both significantly expensive and time-consuming, especially in an assay in which a set of molecular beacons is needed to probe for several different targets, or in a set of assays in which a given target must be probed with a fluorophore of one color in one assay and a different color in another.

SUMMARY

Here, we describe a product, processes or techniques, and methods of using a "Kit of Parts" (KOP) molecular beacon of the present invention that can be quickly and easily assembled, for example, at the lab bench, from a kit consisting of a pair of target-neutral bulk-manufactured labeled oligos (each with either a fluorophore or a quencher attached) together with an inexpensive non-labeled target-specific oligo. Because the labeled oligos are target-neutral, they can be prepared ahead of time and stockpiled for subsequent use; cost and timing efficiencies can be accrued in the process. The target-specific oligo, lacking labels, can be customized for its intended application and quickly and easily manufactured as needed. The target-specific oligo can be combined, for example, at the bench, in a mix and match fashion with stockpiled fluorophore labels of different colors as might be necessary for a set of molecular beacons for the same target used in a suite of assays. In a distinct probe architecture also involving pre-fabricated

components, i.e., Wang's so-called "X-Probe" design architecture, it is noted that a "more than 80%" savings may be achieved compared to a conventional molecular beacon approach. See Wang, J. S. & Wa, D. Y., Simulation-guided DNA probe design for consistently ultraspecific hybridization, Nat. Chem. 7, 545-553,

doi: 10.1038/nchem.2266 (2015) ("Wang"). While the beacons of the present invention have unique structural components and additional steps (such as ligation, as detailed below) not present in Wang's X-Probes, significantly improved functionality is obtained by the KOP molecular beacons of the present invention and a comparable cost savings to that noted in Wang is expected. Moreover, the inventor notes that the decreased turnaround time arising from use of a probe architecture involving pre-fabricated components is also of significance. For example, rather than the current five- to seven- business-day lead time presently required to manufacture particular or customized molecular beacons, commercial synthetic nucleic acids manufacturers (IDT, etc.) can use stockpiled labeled oligos to make KOP molecular beacons instead of classical molecular beacons to significantly increase the speed of manufacture of molecular beacons.

DESCRIPTION OF DRAWINGS

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

Figure 1. Structure and operation of a molecular beacon. Figure from

Wang.

Figure 2. Example DNA sequence and label attachments of a molecular beacon. Figure from Tyagi.

Figure 3. Classically manufactured molecular beacon in the quiescent state.

Figure 4. Classically manufactured molecular beacon in the active state. Figure 5. Quiescent Kit-of-Parts Beacon (before ligation).

Figure 6. Ligated KOP molecular beacon in quiescent state.

Figure 7. Ligated KOP molecular beacon in the active state, hybridized to its target.

Figure 8. Alternate design of a KOP molecular beacon in the quiescent state (before ligation).

Figure 9. Alternate design of a KOP molecular beacon, ligated and in the active state, hybridized to its target.

Figure 10. X-Probe illustration from Zhang.

Figure 11. X-Probe illustration showing strand displacement from

Zhang.

DETAILED DESCRIPTION

The KOP invention shares the use of some nomenclature associated with classically-manufactured molecular beacons. For example, Figure 3 illustrates a classically-manufactured molecular beacon in its unbound, quiescent state. Here the stem regions (also called "domains") of the beacon are labeled in purple, and are notated as s and s', respectively, to connote the fact that they are complementary to one another. In this configuration, the two stems are hybridized together, as illustrated by the pattern of purple dots between them. The loop region between the stem regions, shown in red, is notated as r. A portion of the target oligo to which this beacon is targeted will be complementary to r. The chemical nature of a DNA oligo is that it is directional: one end can be distinguished from the other by details of the chemical linkage of the atoms in the DNA backbone. Following the traditional notational convention, the so-called 3' end of the beacon oligo is denoted with a short half-arrow. See Wikipedia. Directionality (molecular biology),

<https://en.wi kipedia.org/wiki/Directionality _j molecuiar__hiology)> (2016). To that 3' end there is chemically affixed a quencher, shown as a black circle containing a dot. To the other end of the oligo, the so-called 5' end, is affixed a fluorophore, shown as a star. It is noted that while this is the most common and usually -least-expensive orientation for affixing fluorophore and quencher to a molecular beacon, the beacon would work equally well if the orientation were reversed, with fluorophore on the 3' end and quencher on the 5' end. Because the fluorophore and quencher are held in close proximity by the hybridization of the stem regions s and s', the fluorescent nature of the fluorophore is significantly suppressed.

Figure 4 illustrates the classically manufactured molecular beacon in the active state. The intended target DNA oligo is shown in red in the bottom portion of the figure, with dashed lines on its left and right ends illustrating that we are seeing here only a portion of that target. The region of the target to which the beacon attaches itself is labeled as r'. In the figure, the loop region r of the beacon is hybridized to that target r' region, as indicated by the series of red dots denoting the hydrogen bonding of the DNA double helix. The stem regions, s and s are in contrast no longer hybridized. This physically separates the fluorophore and quencher on the ends of the stem regions of the beacon, leading to increased fluorescence. Most significantly because the number of hydrogen bonds in the loop region r exceeds that of the stem region s, thermodynamics favors the transition of a quiescent beacon to an active beacon whenever the beacon encounters its target.

Building on the notations in the previous figures, Figure 5 illustrates essential features of a KOP molecular beacon of the present invention. A KOP beacon is assembled, possibly at a lab bench or at another location outside of a usual nucleic acid synthesis environment, from three separate DNA oligos. The three oligos can be described by listing the sequential functional regions of which they are comprised (as illustrated):

1. Fluorophore oligo: [ sf^" w x y x' >

2. Quencher oligo: [ b' c b a Si >

3. Recognition oligo: [ w' s₂' r s₂ a' >

In this textual notation, a square bracket '[' denotes the 5' end of an oligo, while a pointy bracket '>' denotes the 3' end. Note that a quencher is affixed to the 3' end of the quencher oligo, and a fluorophore is affixed to the 5' end of the fluorophore oligo. Neither the quencher oligo nor the fluorophore oligo contains any regions which are specific to the target of the KOP beacon. By contrast, the recognition oligo is a simple oligonucleotide with no attached chemical moieties. The quencher and fluorophore oligos can thus be bulk-manufactured and stockpiled ahead of time. Only the recognition oligo needs to be manufactured for a specific target, and, lacking the need to affix labels, this can be done relatively quickly and inexpensively.

To assemble a KOP beacon of the present invention, the three oligos are placed in an appropriate buffer solution in approximately equal stoichiometries, but preferably with a small excess of quencher oligos over fluorophore oligos to help maximize the suppression of background fluorescence. If assignments of nucleotides to each of the regions of the oligos has been carried out judiciously during the design process so as to minimize unintentional hybridization (software tools for DNA sequence design are readily available that make this routine), the nature of DNA is such that the oligos will tend to self-assemble into the pattern of hybridization shown in the figure.

If the buffer also contains a DNA ligase, together with the ligase's necessary cofactors, the ligase will then stitch together the nicks in the DNA backbone at the two ligation sites illustrated in Figure 5 and shown in a ligated configuration in Figure 6. See Wikipedia. DNA Ligase, <https:/7en.w¾kipedia. rg wiki/DNA.J¾gase> (2016). This results in a single KOP beacon oligo consisting of the following sequence of regions:

[ si' w x y x' w' s₂' r S2 a' b' c b a si >.

If the presence of the active DNA ligase is compatible with subsequent downstream steps of the assay to which the KOP molecular beacon is being applied, then no further processing is required: an aliquot of the assembled-and-ligated KOP beacon can be simply added to the downstream reaction process. However, if an active ligase is incompatible with downstream processing, the ligase can be deactivated by, e.g., subjecting it to a sufficient thermal shock.

The principles of operation of the KOP beacons of the present invention are as follows. First, they include and improve upon the component parts and functionality provided by classic molecular beacons. The KOP beacons share in common with classic molecular beacons a relatively short stem region (here, sj together with s₂, perhaps some five to eight nucleotides in length overall, much as is the case with classical beacons) to hold the beacon in a closed configuration, with fluorophore adjacent to quencher, until the beacon connects with its target, at which time the recognition region r hybridizing with its target thermodynamically causes the stem to separate. In general terms, the stem separates because the number of hydrogen bonds when r hybridizes to r' exceeds the number of bonds as S] hybridizes with s and s₂ hybridizes with s₂'. The regions a, a', w, and w' are crucial to the self-assembly of the KOP beacon: the self-assembly occurs as a hybridizes with a' and w hybridizes with w '. Each of these regions is long enough, perhaps, for example, some 20 to 25 or so nucleotides, such that at a ligation temperature, the hybridizations involved are effectively stable. Accordingly, the three oligos are kept in place while the ligase has a chance to operate.

The short regions b, b', x, and x' help facilitate the ligation process, as do the short loop regions c andy. Loop regions c andy are minimally three bases long because shorter regions do not allow loopback to occur due to DNA stiffness, and serve simply to allow the quencher or fluorophore oligo to loop back upon itself. Having thus looped back, b, b', x, and x', each perhaps some three to five bases long, holds the end of the fluorophore or quencher oligo adjacent to an end of the recognition oligo, creating the simple DNA-backbone nick circumstance necessary for DNA ligase to perform its function. That is, it is contemplated that for the ligation to work, the presence of a double-stranded section with just a nick in the backbone is required. Here, short regions b, b', x, and x' exist to provide that circumstance; short loop regions c and y exist just to keep short regions b, b', x, and x' connected to their respective starting oligos. These structural and functional features are unique to the present invention as other beacon architectures do not involve strand ligation.

In the ligated KOP beacon, each of the concatenated or linked "assembly regions" shown in Figure 6 in blue and brown a with b and x with w (and a' with V and x' with w') should be long enough (perhaps some 25 or 30 or so nucleotides) such that their melting temperature is higher than any other critical temperatures in the assay to which the KOP beacon is applied. See Wikipedia, Nucleic acid thermodynamics,

(2016). If this guideline is adhered to, then the KOP beacon will behave operationally very much like a classical beacon, as by the time any other (lower) temperature is reached, the KOP beacon will have hybridized into a closed-stem-open-loop structure essentially operationally identical to a classical molecular beacon. The active state of a KOP beacon is shown in Figure 7. Comparisons between Figure 7 and the classical molecular beacon shown in Figure 4 illustrate the distinct architecture and structure of the KOP molecular beacon relative to classical molecular beacons. Further distinctions between the active state of the KOP beacon shown in Figure 7, and the active state of the X- Probe shown in Figure 11, are readily seen.

In one embodiment, the quiescent state of a KOP molecular beacon design variation is illustrated in Figure 8, while its active state is shown in Figure 9. Here, rather than the recognition region being a simple loop comprised of just the r domain, the recognition region is comprised of the r domain hybridized with its complement r'. Adjacent to the r domain is a short "toehold" domain t, perhaps some five to nine nucleotides in length. See Zhang, D. Y. & Winfree, E., Control of DNA strand displacement kinetics using toehold exchange, Journal of the American Chemical Society 131, 17303-17314, doi: 10.1021/ja906987s (2009) ("Zhang"). Connecting the r domain and "toehold" t domain is a linker domain k, perhaps ten or twenty nucleotides in length. Here, both t and r domains are complementary to adjacent portions of the intended target of the probe, and the hybridization of the KOP beacon to the target proceeds by way of strand displacement that is initiated by the hybridization of the t region with its complement t' in the target. See Zhang; Seelig, G., Soloveichik, D., Zhang, D. Y., Winfree, E., Enzyme-free nucleic acid logic circuits. Science 314, 1585- 1588, doi: 10.1126/science. l 132493 (2006). Here, the linker region S plays no role in hybridization; rather, it merely serves to keep the parts of the KOP beacon from separating and coming apart if the KOP beacon is subjected to temperatures beyond the melting points of its various regions, such as would occur during quantitative polymerase chain reaction (qPCR), one of the most common applications of classical molecular beacons. See Wang. Keeping the pieces of the beacon in close proximity in this way maintains their high local concentration, helping to ensure that as the temperature is decreased, the assembly regions will re-hybridize quickly and efficiently in order to create a functional beacon before other temperatures critical to the assay are reached.

In this alternate design, the three oligos from which the KOP beacon can be assembled have the following structure:

1. Fluorophore oligo: [ sf^" w x y x' >

2. Quencher oligo: [ b' c b a Si >

3. Recognition oligo: [ w' s₂' r' k t r s₂ a' >

The domain structure of the corresponding full KOP beacon is as follows:

[ si' w x y x' w' s₂' r' k t r s₂ a' b' c b a Si >

Further, this alternate KOP beacon design can have some additional advantages over the simpler KOP beacon design initially presented. First, the melting temperature of the KOP beacon in the quiescent state here is significantly higher than in the simpler KOP beacon design since it contains significantly more hydrogen bonds. As a result, the quiescent state will more robustly stay closed, and the fluorescence quenched, than in the simpler KOP beacon design. This leads to decreased background fluorescence, and a higher signal-to-noise ratio when the KOP beacon is used in an assay. Also, the amount of single-stranded DNA exposed in the quiescent state whose nucleotide sequence is constrained by being of genomic {i.e., target) origin is less than in the simpler KOP beacon design. This is because in the alternate KOP beacon design, only the short toehold t is exposed, whereas in the simpler KOP beacon design, the longer recognition domain r is exposed. Here it is noted that while the linker domain k is also single stranded, it has no genomic constraints and is entirely synthetic; thus, it can be engineered to avoid inadvertent hybridization. It is the single-stranded regions of a folded DNA oligo which can most easily initiate hybridization, and having a smaller amount of such regions have constraints on their nucleotide assignment facilitates the engineering of an overall KOP beacon which minimizes inadvertent hybridization. The simple KOP molecular beacon design and the alternate KOP molecular beacon design of the present invention differ in several important respects from, for example, the X-Probes described by Wang and illustrated here in Figures 10 and 11, and other dissociative probes. Significantly, X-Probes include four combined oligos; meanwhile, KOP beacons comprise just three combined oligos. X-Probes result from simple hybridizations of their constituent four oligos; meanwhile, KOP beacons result from the ligation of three oligos. Importantly, KOP beacons also include structural and functional features not accounted for, for example, by X-Probe architecture, including regions b, b', x, and x' and short loop regions c a dy, which help facilitate the ligation process.

As a consequence of the structural distinctions between the KOP beacons of the present invention and the X-Probes, X-Probes raised to an elevated temperature more readily disassociate into their constituent parts. And even as X-Probes will tend to reassemble when an elevated temperature causing dissociation is subsequently lowered, it is noted that that reassembly process is stochastic and unreliable in nature and necessitates a carefully controlled annealing process for their creation. Further, such unreliability upon reassembly can adversely affect the suitability of the use of X-Probes in various types of assays. For example, thermal cycling is routine qPCR; thus, X-Probes are ill-suited for that application.

Moreover, unlike the KOP beacons of the present invention, X-Probes are disclosed as having a 5' single-stranded overhang, as shown in Figure 10. See Wang. This overhang will lead to unintended and unacceptable side-products during a PCR amplification process.

Here, the inventor notes that Wang proposes chemical alteration of the 3' end (of their P strand) to prevent unwanted extension by polymerase activity. By contrast, the present invention instead overcomes problems associated with unwanted polymerase activity extensions by reversing the direction of the DNA strands in their design architecture.

Moreover, it is important to understand that with X-Probes, the four constituent strands necessarily come together and hybridize only as part of the ultimate reaction mixture to which they are applied. Though some amount of pre-mixing and pre-assembly of the constituents may be possible so long as inappropriate thermal shocks are not subsequently applied, such gentle thermal treatment may or may not be possible, according to the particular assay to which they applied. In contrast, KOP molecular beacons are a pre-assembled and pre-ligated combination of three oligos. By the time they are applied to an assay, they are a cohesive single DNA oligo. So long as the constituent nucleic acid hybridization events of the assay occur at temperatures below the melting temperature of the assembly regions ab-a'V and xw-x'w' of the probe, a KOP molecular beacon can be used in virtually any assay in which a classical molecular beacon would otherwise be applicable. The same cannot be said of X-Probes.

KOP beacons have some constraints on their DNA sequence design. In this context, DNA sequence design is the act of assigning DNA nucleotides to regions of the constituent DNA oligos with intent of realizing an intended hybridization pattern, and only that pattern, when the oligos are synthesized according to that design and allowed to come together. As has been mentioned, the labeled oligos in KOP beacons are designed to contain no, or avoid including, a target-specific region as they are not intended to hybridize to the beacon or probe target. This lack of hybridization potential is the property that allows the labeled oligo components to be bulk-manufactured in advance and stockpiled. Optimally, the labeled oligos of the present invention KOP beacons, should not hybridize at all with any target in a significant way, even inadvertently. In this sense, the labeled oligos should be universal. Clearly, this is a goal which is, in theory at least, likely ultimately unobtainable. Accordingly, and for example, a given stockpile of labeled oligos for a KOP beacon should not be used with targets containing a significant subset of the DNA sequences found in the assembly regions of the beacon. However, as a practical matter, this is not a cause for concern.

If the domains in the labeled oligos are of sufficient length so as to encompass a large enough sequence design space, and so long as sufficient

computational effort is expended with DNA sequence design tools to explore that space well, one can design a single pair of labeled oligos for a KOP molecular beacon that will work well with nearly any target, including genomic ones, which are far from random due to their need to embody biological function. In the rare circumstance that a designed pair of labeled oligos is found to interact poorly with a novel target, a repeat of the DNA sequence design run with that target as context is expected to eliminate the immediate problem. While this new run will regrettably result in the need to

manufacture a second stockpile of labeled oligos, with two stockpiles in hand, chances are increased that any new targets will work well with one or the other. Ultimately, a small handful of labeled oligo designs ought to provide a working stock that is indeed is truly universal, in that one of the designs in the handful will work well with virtually any target.

In some embodiments of the present invention, more than three starting oligos may be used in accordance with the inventive principles described herein.

In one such embodiment, for example, one could start with a larger number of starting oligos so long as the sequences self-assemble into double-stranded helices with nicks so that ligation can stitch the oligos together.

In one embodiment, more than three starting oligos may be used to form a beacon of the present invention that advantageously additionally comprises a short oligo having a biotin moiety attached to one of the internal T bases (commercially available). Such an oligo may then be used as part of the present invention to stick or bind the strand having an attached biotin moiety to anything coated with streptavidin. It is contemplated that streptavidin-coated magnetic beads could then be used to physically separate such beacons of the present invention (perhaps with their targets). This embodiment of the present invention would thus advantageously combine fluorescence quantitation and physical separation technologies. Nonetheless, it is noted that in those contexts or experiments where physical separation of the beacon (and perhaps its target) are not needed, biotinylated oligos may be omitted and substituted with plain oligos with the same sequence.

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A molecular beacon comprising at least one prefabricated labeled oligo comprising a self-assembled ligation site region and a loop region.

2. The molecular beacon of claim 1, further comprising a recognition oligo.

3. The molecular beacon of claim 2, wherein either the prefabricated labeled oligo further comprises a first half of a self-assembly region or the recognition oligo comprises a second half of the self-assembly region that is complementary to the first half of the self-assembly region.

4. The molecular beacon of claim 2, wherein the recognition oligo comprises a toehold region and a linker region.

5. The molecular beacon of claim 1, comprising two prefabricated labeled oligos, each comprising a self-assembled ligation site region and loop region.

6. The molecular beacon of claim 5, further comprising a recognition oligo.

7. The molecular beacon of claim 6, wherein the prefabricated labeled oligos each comprise a first half of a self-assembly region and the recognition oligo comprises a second half of the self-assembly region that is complementary to at least one first half of the self-assembly region.

8. The molecular beacon of claim 6, wherein the recognition oligo comprises a toehold region and a linker region.

9. A molecular beacon ligation product comprising two prefabricated labeled oligos and a third recognition oligo.

10. The product of claim 9, wherein at least one of the prefabricated labeled oligos comprises a first half of a self-assembly region and the recognition oligo comprises a second half of the self-assembly region that is complementary to the first half of the self-assembly region.

11. The product of claim 9, wherein at least one prefabricated labeled oligo comprises a self-assembled ligation site region and a loop region.

12. The product of claim 9, wherein each of the prefabricated labeled oligos comprises a self-assembled ligation site region and a loop region.

13. The product of claim 9, wherein the recognition oligo comprises a toehold region and a linker region.

14. The product of claim 9, wherein at least one of the prefabricated labeled oligos comprises a first half of a self-assembly region and the recognition oligo comprises a second half of the self-assembly region that is complementary to the first half of the self-assembly region.

15. A molecular beacon comprising one or more prefabricated labeled oligos and a recognition oligo comprising a toehold region and a linker region.

16. The molecular beacon of claim 15, wherein at least one prefabricated labeled oligo comprises a self-assembled ligation site region and loop region.

17. The molecular beacon of claim 15, comprising two prefabricated labeled oligos.

18. The molecular beacon of claim 15, wherein at least one of the prefabricated labeled oligos comprises a first half of a self-assembly region and the recognition oligo comprises a second half of the self-assembly region that is complementary to the first half of the self-assembly region.

19. A target hybridized molecular beacon oligo comprising hybridized self-assembly regions on either side of a target.

20. The oligo of claim 19, wherein the hybridized self-assembly regions are associated with each of two prefabricated labeled oligos and a recognition oligo.

21. The oligo of claim 19, wherein at least one prefabricated labeled oligo comprises a self-assembled ligation site region and a loop region.

22. The oligo of claim 19, further comprising a toehold region and a linker region.

23. A molecular beacon oligo in an active state configuration comprising hybridized self-assembly regions on either side of the target.

24. The oligo of claim 23, wherein the hybridized self-assembly regions are associated with each of two prefabricated labeled oligos and a recognition oligo.

25. The oligo of claim 23, wherein at least one prefabricated labeled oligo comprises a self-assembled ligation site region and a loop region.

26. The oligo of claim 23, further comprising a toehold region and a linker region.

27. A molecular beacon in a quiescent state configuration comprising two linked self-assembly regions, wherein each linked self-assembly region is positioned opposite to the other.

28. The molecular beacon of claim 27, wherein one of the two linked self- assembly regions comprises a first self-assembly region provided on a prefabricated labeled oligo and a second self-assembly region on a recognition oligo that is complementary to the first half of the self-assembly region.

29. The molecular beacon of claim 28, wherein the recognition oligo further comprises a toehold region and a linker region.

30. A method for making a molecular beacon comprising: forming at least one labeled oligo;

forming a recognition oligo; and

joining the at least one labeled oligo to the recognition oligo by ligation.

31. The method of claim 30, further comprising:

including a toehold region and a linker region on the recognition oligo.

32. The method of claim 30, further comprising:

preparing the labeled oligo for self-assembly of a ligation site region and loop region.

33. The method of claim 30, wherein the molecular beacon self-assembles in the presence of ligase.

34. A method of using a molecular beacon ligation product of any of claims 1, 9, 15, 19, 23, and 27 for detection of single-nucleotide variants at low variant allele frequency in one of DNA and RNA.

35. A method of using a molecular beacon ligation product of any of claims 1, 9, 15, 19, 23, and 27 for detection of single-nucleotide variants at low variant allele frequency in one of DNA and RNA for detection of at least one of early cancer, subtyping of infectious disease, and identification of drug-resistant infectious disease.

36. A method of using a molecular beacon ligation product of any of claims 1, 9, 15, 19, 23, and 27 for assays involving DNA or RNA hybridization.

37. A method of using a molecular beacon ligation product of any of claims 1, 9, 15, 19, 23, and 27 in primer-mediated enzymatic amplification assays.