WO2019012481A2

WO2019012481A2 - Split g-quadruplexes for capture and detection of nucleic acids

Info

Publication number: WO2019012481A2
Application number: PCT/IB2018/055166
Authority: WO
Inventors: John Katz
Original assignee: John Katz
Priority date: 2017-07-12
Filing date: 2018-07-12
Publication date: 2019-01-17
Also published as: BR112020000638A2; WO2019012481A3

Abstract

Methods of using split G-quadruplexes associated with functional tags for associating said tags to target nucleic acids. Methods include use of split G- quadruplexes associated with detection tags for the detection of target nucleic acids, and use of split G- quadruplexes associated with capture tags for detection or capture of target nucleic acids.

Description

SPLIT G-QUADRUPLEXES FOR CAPTURE AND DETECTION

OF NUCLEIC ACIDS

[001] The invention relates to methods of associating functional tags to target nucleic acids, and uses thereof, including the detection of target nucleic acids, and the capture of target nucleic acids.

BACKGROUND OF THE INVENTION

[002] G-quadruplexes are structures formed in nucleic acids by sequences that are rich in guanine. Four guanine bases can associate through Hoogsteen hydrogen bonding to form a square planar structure called a guanine tetrad, and two or more guanine tetrads can stack on one another to form a G-quadruplex . The quadruplex structure is further stabilized by the presence of a cation, which sits in a central channel between each pair of tetrads. They can be formed of DNA or RNA (or other nucleic acids); and they typically form from one strand

(intramolecular), two strands (bimolecular) , three strands ( trimolecular) , or four stands ( tetramolecular) of nucleic acid. G-quadruplexes with 4 G-rich sequences aligned in the same 5'-to-3' direction are termed parallel; G-quadruplexes with 2 G-rich sequences aligned 5'-to-3' and 2 G-rich sequences aligned 3'-to-5' direction are termed anti-parallel; and G quadruplexes with either 3 G-rich sequences aligned 5'-to-3' and 1 G- rich sequence aligned 3'-to-5', or 1 G-rich sequence aligned 5'-to-3' and 3 G-rich sequences aligned 3'-to-5', are termed mixed or hybrids . [003] Bases intervening the G-rich sequences are required for proper folding of the G-quadruplex, especially for bimolecular and intramolecular structures. Upon G-quadruplex formation, these bases reside in loop regions, and G-quadruplexes of different topologies have loops in different configurations. For example, quadruplexes in a parallel topology will have loops in a propeller configuration (positioned to the side of the quadruplex) , whereas quadruplexes in an anti-parallel topology will have loops in a lateral configuration

(joining adjacent G-rich sequences), or in both lateral configuration and diagonal configuration (joining diagonally opposite G-rich sequences) . Further, studies have developed the consensus sequence G₃₊Ni-₇₊G₃₊Ni-₇₊G₃₊Ni- ₇₊G₃ (where N is any base including guanine) to identify putative G-quadruplexes, with G₃ representing the G-rich sequences, and Ni_₇ representing the intervening bases in the loop regions. Examples of published and theoretical G-quadruplexes are found on the G4RNA Database

(http://scottgroup.med.usherbrooke.ca) and GregList (G- quadruplex Regulated Genes List)

(http://tubic.tju.edu.cn/greglist) , respectively.

[004] G-quadruplex sequence is present in the genomes of a variety of organisms. In humans, genome-wide surveys have identified >376,000 Putative Quadruplex Sequences, although not all of these probably form in vivo. Some sequences are found in human telomeres with the DNA repeat d(GGTTAG)_n. The formation of quadruplexes in telomeres has been shown to decrease the activity of the enzyme telomerase, which is responsible for maintaining the length of telomeres, and is involved in around 85% of all cancers. Other sequences are found in promoter regions of genes, including the proto-oncogenes c-myc, k- ras, c-kit, Bcl-2, and VEGF.

[005] In addition to cations, other molecules have been identified that influence formation of or bind to G- quadruplexes . Some molecules induce formation of G- quadruplexes , including the DNA binding protein RAPl, the crowding agent polyethylene glycol, and the ionic liquid guanidinium tris (pentafluoroethyl ) trifluorophosphate

(Gua-IL) . Other molecules are capable of binding G- quadruplexes , including the helicase BLM, the DNA binding protein RAPl, the engineered zinc finger protein Gql, the G-quadruplex-specific antibody 1H6, and the small molecules hemin, NMM, TMPyP4, and telomestatin . Interestingly, a subset of these molecules also stabilizes formation of G-quadruplexes (ex. Gua-IL, TMPyP4, and telomestatin) . The G-quadruplex Ligands Database (http://www.g41db.org) lists hundreds of molecules that influence or bind G-quadruplexes.

Catalytic G-Quadruplexes

[006] G-quadruplexes have been isolated from random

DNA libraries using aptamer selection methodology SELEX and the molecule NMM - a transition state analog of heme, an enzyme cofactor found in peroxidases and other enzymes. G-quadruplexes bind NMM with micromolar affinity

(via end-stacking) , and interestingly, bind hemin (an oxidized form of heme) with micromolar affinity too. With addition of oxidizing agent H₂O₂, the G-quarduplex-hemin complex is capable of oxidizing a variety of substrates, including colorimetric and chromogenic substrates (ex. DAB, ABTS) - and chemiluminescent substrates (ex. luminol) - used in peroxidase assays. The G-quadruplex- hemin complex is approximately two orders of magnitude more reactive than hemin alone in catalyzing peroxidase reactions .

[007] Hence, G-quadruplexes are considered to be DNA enzymes. Studies have shown that G-quaduplex-hemin complexes are less active than horseradish peroxidase

(HRP) but more active than the enzyme catalase. Interestingly, the G-quadruplex-hemin complex displays a broader range of substrate specificity than HRP, and a higher rate of self-inactivation than HRP - likely because of a more exposed active site. Some studies have shown G-quadruplex activity dependent on ions, buffers, pH, and surfactants - as well as activity enhancement agents such as adenosine triphosphate and spermidine. Other studies have shown G-quadruplex activity dependent on loop size, flanking sequence, and topology. G- quadruplex-hemin complexes have many advantages in comparison to HRP; however, weaker peroxidase activity and higher inactivation rate have hindered G-quadruplex use as HRP replacements.

Split G-Quadruplexes

[008] Split G-quadruplexes are engineered G- quadruplexes that are used to detect nucleic acids via their inherent catalytic activity. These molecules were first designed by dividing the G-quadruplex sequence into an upstream sequence and a downstream sequence, and attaching target-binding arms to the upstream and downstream sequences. Hence, split G-quadruplexes comprise two oligonucleotide strands - each with a partial G-quadruplex sequence and a target-binding arm. The target-binding arms are single stranded, and designed to bind single stranded target nucleic acid, for example, designed with sequence complementary to the target nucleic acid, and thus capable of binding said nucleic acid. Accordingly, in the presence of the target, the split G-quadruplex binds and its G-quadruplex assembles and becomes competent to catalyze its peroxidase reaction .

[009] The use of split G-quadruplexes with peroxidase activity to detect nucleic acids is interesting, especially with molecules demonstrating high binding specificity (using short target binding arms) . However, studies have observed low target sensitivity (ex. ΙΟηΜ- to-lmM using colorimetric substrates) in comparison to HRP assays (ex. 0. lpM-to-100pM) . The low target sensitivity probably reflects the aforementioned limitations of (i) weaker peroxidase activity and (ii) higher inactivation rate in comparison to HRP. As with G- quadruplexes , these limitations have similarly hindered use of split G-quadruplexes as nucleic acid detection agents .

SUMMARY OF THE INVENTION

[010] The present invention describes methods and reagents for associating tags to nucleic acids, the method comprising associating a tag to a split G- quadruplex, and binding a split G-quadruplex to a nucleic acid. In some aspects, the tag is a capture tag, which can be used to capture a split G-quadruplex, and accordingly, capture a nucleic acid bound by the split G- quadruplex. In other aspects, the tag is a detection tag, which can be used to detect a split G-quadruplex, and accordingly, detect a nucleic acid bound by the split G- quadruplex .

[Oil] Tags are nucleic acid modifications that impart characteristic features to the nucleic acid, such as the ability to be captured, detected, targeted, or crosslinked. Tags are known in the art, commonly sold by oligonucleotide manufacturers, and can be bound or incorporated into nucleic acids, such as attachment chemistries, fluorophores , detectable enzymes, detectable particles, and nucleotide analogs.

[012] In some aspects, the disclosure provides a kit for capturing or detecting nucleic acids comprising a split G-quadruplex and an associated tag used to capture or detect said split G-quadruplex. In other aspects, the disclosure provides an apparatus for capturing or detecting nucleic acids comprising a split G-quadruplex and an associated tag used to capture or detect said split G-quadruplex.

BRIEF DESCRIPTION OF DRAWINGS

[013] Figure 1 is an illustration of six embodiments of the disclosed invention for the capture or detection of target nucleic acids (dashed line) utilizing split G- quadruplexes associated with capture tags (C) or detection tags (D) . In the upper box, an antibody (Ab) , a split G-quadruplex (SQ) , and a split-G-quadruplexes with capture arms (SQB) are illustrated. Some embodiments feature a capture nucleic acid (solid line) . The capture tag of the nucleic acid used to bind the solid surface

(large black rectangle) is not illustrated. DESCRIPTION OF THE INVENTION

[014] The present invention describes a surprisingly stable interaction between split G-quadruplexes and target nucleic acids. The high binding affinity of split G-quadruplexes (for target nucleic acids) does not require peroxidase activity - and likely reflects formation of the G-quadruplex upon target binding, which physically links the two target binding arms of the split G-quadruplex. The combination of high binding affinity and high binding specificity are features shared with antibodies, which are used to associate (or bridge) tags to antigens. Accordingly, the present invention discloses the use of split G-quadruplexes to associate tags to nucleic acids, for example, in methodologies to capture nucleic acids, and methodologies to detect nucleic acids. The present invention also discloses the use of split G-quadruplexes (in place of antibodies) in several antibody methodologies adapted for nucleic acids, including purification, precipitation, targeting, crosslinking, and modification of nucleic acids - as well as kits and apparatuses based on said methodologies.

Split G-Quadruplexes

[015] Split G-quadruplexes are engineered G- quadruplexes designed to bind target sequences in target nucleic acids, and upon binding, assemble into G- quadruplexes . These molecules can comprise two, three, or four strands of nucleic acid, with (i) each strand containing a partial sequence of a G-quadruplex, and (ii) two or more strands with single-stranded target-binding arms, each capable of binding part of a single-stranded target sequence of the target nucleic acid. In one embodiment, target-binding arms are designed with sequence capable of binding the target sequence, for example, by hybridization. In another embodiment, target- binding arms are designed with sequence complementary to the target sequence, and thus are capable of binding the target sequence by hybridization. In a preferred embodiment, the sum of the partial sequences of the split G-quadruplex is a G-quadruplex, and the sum of the target-binding arm sequences is a sequence complementary to the target sequence.

[016] A split G-quadruplex of x strands (where x=2,

3, or 4) can be designed by (i) dividing the G-quadruplex sequence into x partial sequences (ex. for x=2, divided into an upstream and downstream sequence, ex. containing 1, 2, or 3 G-rich sequences); (ii) determining the complementary sequence of the target sequence, dividing the complementary sequence into y partial sequences

(where y=2, 3, or 4); and (iii) placing a partial complementary target sequence on each strand of the split G-quadruplex. The partial complementary target sequences

(herein called the target binding arms) can be placed on the 5' -end, 3' -end, or in an internal position of the partial G-quadruplex sequence. For example, a split G- quadruplex of two strands can have a first strand with either (i) the 5' -end of the target binding arm (with upstream complementary target sequence) attached to the 3' -end of the upstream G-quadruplex sequence, or preferentially (ii) the 3' -end of the target binding arm

(with upstream complementary target sequence) attached to the 5' -end of the upstream G-quadruplex sequence; and a second strand with either (iii) the 5' -end of the downstream G-quadruplex sequence attached to the 3' -end of the target binding arm (with downstream complementary target sequence), or preferentially (iv) the 3' -end of the downstream G-quadruplex sequence attached to the 5'- end of the target binding arm (with downstream complementary target sequence) . In another embodiment, a split G-quadruplex strand is attached to two or more target binding arms. In a preferred embodiment, the target-binding arms are attached to the partial G- quadruplex sequences via a linker or spacer - such as Phosphoramidite C3, Hexanediol, and 1 ' , 2 ' -Dideoxyribose, and preferentially via the spacers Triethylene Glycol or Hexa-Ethyleneglycol .

[017] Split G-quadruplex strands can be designed with different combinations of partial G-quadruplex sequences. For example, a split G-quadruplex of two strands can have a first strand with 3 G-rich sequences and 2 loops of a G-quadruplex, and a second strand with 1 G-rich sequence of a G-quadruplex. In a preferred embodiment, the first strand and second strands each comprise 2 G-rich sequences and 1 loop of a G-quadruplex. In another embodiment, the first strand comprises the G-quadruplex sequence G3₊Ni_₇₊G₃₊Ni_₇₊G3, and the second strand comprises the G-quadruplex sequence G3, wherein N is any base including guanine. In a preferred embodiment, the first and second strand sequences each comprise the G- quadruplex sequence G₃₊Ni_₇₊G₃, wherein N is any base including guanine. In another preferred embodiment, the first strand comprises the G-quadruplex sequence GGGTAGGG, and the second strand comprises the G- quadruplex sequence GGGTTGGG. And split G-quadruplexes of three and fours strands can be designed by dividing (splitting) the above G-rich sequences, such as one 2 G- rich and two 1 G-rich strands for a three strand G- quadruplex, and four 1 G-rich strands for a four strand quadruplex .

[018] A target sequence can be a single continuous sequence in the target nucleic acid. Accordingly, in one embodiment, the target binding arms of the split G- quadruplex are designed to bind two flanking regions

(parts) of said continuous target sequence. A target sequence can also be two or more physically separate sequences (ex. parts, separated by non-target sequence) in the target nucleic acid. Accordingly, in another embodiment, the target-binding arms of the split G- quadruplex are designed to bind two or more physically separate sequences of the target sequence. In another embodiment, one or more target binding arms are made short in length, so in the chosen conditions and temperature for binding, the split G-quadruplex hybridizes to perfect target sequences and not alternative sequences, including sequences with nucleotide substitutions, such as single nucleotide polymorphisms (SNPs) . For example, the short length can be selected by (1) choosing a temperature for operation of the split G-quadruplex (ex. 25°C) , and (2) designing a pair of target binding arms, with one arm having a melting temperature (Tm) equal or greater than the operation temperature (ex. 40°C), and one arm having a Tm equal of less than the operation temperature (ex. 20°C), wherein a nucleotide substitution in the target significantly lowers the Tm (ex. <0°C) . Such a design permits the use of the split G-quadruplexes to capture or detect perfect target sequences, or alternatively, capture or detect sequences with nucleotide substitutions, including SNPs .

[019] In one embodiment, split G-quadruplexes are used with molecules that influence the formation of G- quadruplexes , or bind to G-quadruplexes. In a preferred embodiment, split G-quadruplexes are used with molecules that induce the formation of G-quadruplexes, such as cations (ex. Na⁺, K⁺, NH₄ ⁺) , the DNA binding protein RAPl, the crowding agent polyethylene glycol, and the ionic liquid guanidinium tris (pentafluoroethyl ) trifluorophosphate (Gua-IL) . In another preferred embodiment, split G-quadruplexes are used with molecules that stabilize the formation of G- quadruplexes , such as Gua-IL, TMPyP4, and telomestatin . And in another preferred embodiment, split G-quadruplexes are used with molecules that promote the catalytic activity of G-quadruplexes, such as ATP.

[020] Melting temperature studies have found G- quadruplexes with parallel topologies to be more stable than G-quadruplexes with anti-parallel topologies. In the present invention, split G-quadruplexes with greater stability - ex. with parallel topology - are expected to bind targets more stably, and can be used in methods requiring improved target binding. Similarly, split G- quadruplexes with weaker stability - ex. with anti- parallel topology - are expected to bind targets more weakly, and can be used in methods requiring reduced target binding (ex. to reduce background or improve specificity) . G-quadruplexes can be designed or treated

(ex. with chemicals) to assume different topologies, such as a parallel topology, an anti-parallel topology, or a hybrid topology. For example, G-quadruplexes designed with short loops favor parallel topologies, and G- quadruplexes with long loops favor anti-parallel topologies. For example, G-quadruplexes treated with K⁺ favor parallel topologies, and G-quadruplexes treated with Na⁺ favor anti-parallel topologies. In the present invention, split G-quadruplexes can be similarly designed or treated to favor parallel topologies or favor anti- parallel topologies.

Tags

[021] Tags are nucleic acid modifications that can be associated - ex. bound or incorporated - to nucleic acids, and similarly, can be associated - ex. bound or incorporated - to split G-quadruplexes. Tags are functional, and their association to a nucleic acid imparts their function to the nucleic acid. For example, a tag that can be detected (ex. a detection tag), bound or incorporated to a nucleic acid, permits said nucleic acid to be detected. For example, a tag that can be captured (ex. a capture tag), bound or incorporated to a nucleic acid, permits said nucleic acid to be captured. For example, a tag that can be targeted (ex. a target tag) , bound or incorporated to a nucleic acid, permits said nucleic acid to be targeted. For example, a tag that can be crosslinked (ex. a crosslinked tag), bound or incorporated to a nucleic acid, permits said nucleic acid to be crosslinked.

[022] Tags are usually associated to nucleic acids by binding or incorporation to the nucleic acid (ex. during nucleic acid synthesis) . Tags and nucleic acid modifications are known in the art, and many are available from oligonucleotide manufacturers. Examples of detection tags include fluorophores , quenchers, phosphoylation, detectable enzymes (horseradish peroxidase), dyes, reactants (ex. acrydite) , detectable particles, and nucleotide analogs (ex. fluorescent, radiolabeled) ; and include acrydite, Cyanine dyes, 6-FAM, Fluorescein-dT, HEX, JOE, Lightcycler 640, ROX, SYBR Green, TAMRA, TET, Texas Red-X, Alexa Fluor dyes, Rhodamine dyes, WellRED dyes, Black Hole quenchers, DABCYL, and 2-aminopurine . Examples of capture tags include attachment chemistries, binding chemistries, phosphorylation, antibody antigens, antibodies, nucleotide analogs (ex. that are antibody antigens), and nucleotide sequences (ex. that hybridize to other nucleic acids); and include adenylation, alkyne modifiers (ex. click reaction) , amino modifiers, avidin, azide, biotin, cholesterol, digoxigenin (DIG), 2 , 4-dinitrophenol (DNP) , and thiol modifiers. Examples of target tags include cholesterol and phosphorylation. Examples of crosslink tags include 5-bromo-deoxyuridine. Other tags, such as nucleotide analogs (which include modified nucleotides, ex. nucleotides with modified nucleobases ) , have members with functions similar to the aforementioned tags, such as 2-aminopurine (detection) and 5-bromo-2 ' -deoxyuridine (BrdU) ( crosslinking) .

[023] Detection tags are nucleic acid modifications that can be detected, for example, by senses (ex. visually), or by use of assays or equipment (ex. measuring the presence, amount, or functional activity of the detection tag) . Commonly used detection tags are fluorophores and quenchers, which can be detected by fluorometer or microscope. Other detection tags that can be used include phosphorylation and nucleotide analogs (ex. radiolabeled and detected by scintillation counter or autoradioagraphy (ex. film)), detectable enzymes (ex. horseradish peroxidase), detectable particles (ex. colloidal gold and colored latex), and BrdU (ex. crosslinking the labeled nucleic acid (ex. the split G- quadruplex) with the target nucleic acid, and then measuring the presence of the two crosslinked nucleic acids by electrophoresis or chromatography) .

[024] Capture tags are nucleic acid modifications that bind, or are capable of binding, to specific molecules - herein called capture targets. Some capture tags rely on high-affinity non-covalent bonds for capture target binding, such as biotin (binding to avidin) and digoxigenin and 2 , 4-dinitrophenol (binding to anti-DIG and anti-DNP antibodies, respectively) . Other capture tags rely on covalent bonds for capture target binding, which often require chemical treatment to activate reactive groups on the capture tag (or capture target) , such as amino modifiers, alkyne modifiers, and thiol modifiers. Examples of capture targets include nucleic acids (including the herein capture nucleic acids), molecules than can be detected (ex. dyes and enzymes), molecules capable of binding other molecules (ex. antigens and antibodies), and solid surfaces.

[025] Target tags such as cholesterol and phosphorylation can be used to facilitate nucleic acid uptake into cells. Target tags can be similarly incorporated into a split G-quadruplex, for example, to facilitate its uptake into cells, and to facilitate the uptake of target nucleic acids bound by the split G- quadruplex. Such split G-quadruplexes can also be associated with DNA regulatory molecules - ex. enzymes, nucleases, transcription factors, enzyme inhibitors, enzymes subtrates, enzymes catalysts, etc. - in order to target said molecules to specific target nucleic acids within cells, for example, for use in gene regulation, protein expression via RNA regulation, or anti-viral or anti-bacterial therapy. Crosslink tags such as BrdU are used to crosslink target nucleic acids to other nucleic acids or proteins. Crosslink tags can similarly be incorporated into a split G-quadruplex in order to crosslink it to other nucleic acids, or to other proteins, which then can be associated to a target nucleic acid by binding said split G-quadruplex to the target nucleic acid.

[026] Split G-quadruplexes can contain one or more tags - bound or incorporated in one, two, three, or four of its strands. The tags can be placed on the 5' -end, 3'- end, or the middle of a strand, and different tags can be placed on one strand or different strands of the split G- quadruplex. In one embodiment, tags are placed distant from the partial G-quadruplex, for example, one strand can have a tag bound or incorporated to the 5' -end of a target-binding arm, and the 3' -end of the target-binding arm attached to the 5' -end of the partial G-quadruplex sequence. Such a configuration may be desired if the capture tag - or capture target (ex. solid surface) attached to the capture tag - can interfere with the assembly of the split G-quadruplex. In another embodiment, tags are placed proximal to the partial G- quadruplex, for example, one strand can have the 3' -end of the target-binding arm attached to the 5' -end of the partial G-quadruplex sequence, and the 3' -end of the partial G-quadruplex sequence bound or incorporated with the tag. Such a configuration may be desired if multiple capture tags (ex. biotins) can bind a single target molecule (ex. avidin) , which can result in multiple G- quadruplex strands being captured next to one another, and assembling artificially into a G-quadruplex (without target binding arm binding to the target nucleic acid) . Placing the capture tag proximal to the partial G- quadruplex effectively locates said G-quadruplex sequence to a single binding site on the capture target, and reduces the probability that said sequence can interact with other G-quadruplexes bound to other binding sites on the same or neighboring capture targets. Additional binding sites can be blocked by using capture tags that can bind multiple binding sites on the same or neighboring capture targets, for example, a commercially available capture tags with two biotins (attached by long linkers) .

Capture Arms

[027] Additional target-binding arms - capable of binding other nucleic acid sequences - can be added to split G-quadruplexes to capture other target nucleic acids. These additional arms function as sequence- dependent capture tags, capable of binding nucleic acids as their capture targets, and can be designed similarly to, or different from, the aforementioned target-binding arms. Accordingly, herein, different nomenclature is utilized, where these additional target-binding arms are called capture arms, which bind target sequences called capture sequences, that are present in target nucleic acids called capture nucleic acids. In one embodiment, capture arms are designed similarly to target-binding arms, wherein each strand of the split G-quadruplex contains a single-stranded capture arm, which is capable of binding part of the single-stranded capture sequence of the capture nucleic acid. In a preferred embodiment, one or more capture arms are made short in length, so in the chosen conditions and temperature for binding, the split G-quadruplex hybridizes to perfect capture sequences and not alternative sequences, including sequences with nucleotide substitutions, such as SNPs . In another embodiment, capture arms are designed differently to target-binding arms, wherein one strand of the split G-quadruplex contains a capture arm, which is capable of binding the entire capture sequence of the capture nucleic acid. And in another embodiment, the sum of the capture arm sequences is a sequence complementary to the capture sequence.

[028] A split G-quadruplex of n strands (where n=2,

3, or 4) and x capture arms (where x=l, 2, 3, or 4, and x ≤ n) can be designed by (i) dividing the G-quadruplex sequence into n partial sequences (ex. for n=2, divided into an upstream and downstream sequence, ex. containing 1, 2, or 3 G-rich sequences), and placing a partial sequence on each strand of the split G-quadruplex; (ii) determining the complementary sequence of the target sequence, dividing the complementary sequence into n partial sequences (ex. for n=2, divided into an upstream and downstream sequence) , and placing a partial complementary target sequence on each strand of the split G-quadruplex; and (iii) determining the complementary sequence of the capture sequence, dividing the complementary sequence into x partial sequences (ex. for x=2, divided into an upstream and downstream sequence), and placing a partial complementary capture sequence on one or more strands of the split G-quadruplex. For example, a split G-quadruplex of two strands can have a first strand with the 3' -end of the target binding arm (with upstream complementary target sequence) attached to the 5' -end of the upstream G-quadruplex sequence, and the 3' -end of the upstream G-quadruplex sequence attached to the 5' -end of the capture arm (with downstream complementary capture sequence); and the 3' -end of the capture arm (with upstream complementary capture sequence) attached to the 5' -end of the downstream G- quadruplex sequence, and 3' -end of the downstream G- quadruplex sequence attached to the 5' -end of the target- binding arm (with downstream complementary target sequence) . In a preferred embodiment, the target-binding arms and the capture arms are attached to the partial G- quadruplex sequences via a linker or spacer - such as Phosphoramidite C3, Hexanediol, and 1 ' , 2 ' -Dideoxyribose, and preferentially via the spacers Triethylene Glycol or Hexa-Ethyleneglycol .

[029] Capture nucleic acids can be bound or incorporated with functional tags; for example, detection tags, capture tags, target tags, or crosslinked tags. Accordingly, capture nucleic acids can be used to associate functional tags to target nucleic acids; for example (i) a functional tag is associated to a capture nucleic acid, (ii) said capture nucleic acid is bound to the capture arms of a split G-quadruplex, and (iii) the target binding arms of said G-quadruplex is bound to a target nucleic acid. Such methods permit the use of capture nucleic acids with split G-quadruplexes to detect, capture, target, or crosslink target nucleic acids. For example, methods are described below for the detection of target nucleic acids using a capture nucleic acid with a detection tag, and split G-quadruplexes . For example, methods are described below for the capture of target nucleic acids onto a solid surface using capture nucleic acids with capture tags, and split G- quadruplexes .

Methods to Detect Nucleic Acids

[030] In one embodiment, target nucleic acids are detected by (i) associating a detection tag to a split G- quadruplex, (ii) binding said split G-quadruplex to the target nucleic acid, and (iii) detecting the detection tag with a method known in the art. Examples of detection tags that can be used include fluorophores , quenchers, phosphoylation, and nucleotide analogs. In another embodiment, target nucleic acids are detected by (i) binding a split G-quadruplex to a target nucleic acid,

(ii) associating a detection tag to the split G- quadruplex, and (iii) detecting the detection tag with a method known in the art. Examples of detection tags that can be used include phosphorylation and nucleotide analogs. In a preferred embodiment, the target nucleic acid is bound to a solid surface - either (i) before the target nucleic acid is bound to the split G-quadruplex; or (ii) after the target nucleic acid is bound to the split G-quadruplex, but before detection of the detection tag - permitting washing of said target nucleic acid and removal of unbound detection tags before detection (of bound detection tags) . An illustration of a target nucleic acid associated to a detection tag using said methods and a solid surface (i.e. preferred embodiment) is shown in Figure 1 Embodiment 1.

[031] A second approach to detect target nucleic acids uses capture tags to bind capture targets that can be detected, or capture targets that can bind detectable molecules. Examples of detectable capture targets (and detectable molecules) include detection tags and immunoassay labels, and detectable enzymes, fluorophores , detectable particles, and radiolabeled molecules. Examples of detectable enzymes include enzymes that catalyze chromogenic or chemiluminescent reactions, such as alkaline phosphatase (AP) , horseradish peroxidase

(HRP) , beta-galactosidase (b-gal), and luciferase (LUC), and DNA enzymes such as Catalytic G-Quadruplexes . Examples of detectable fluorophores include fluorescein isothiocyanate (FITC) and tetramethylrhodamine (TRITC) . Examples of detectable particles include colloidal gold, colored or fluorescent latex, and paramagnetic latex particles. And examples of detectable radiolabeled molecules include antibodies and antigens labeled with 125-1 or 3-H.

[032] In one embodiment, target nucleic acids are detected using capture tags and capture targets by (i) associating a capture tag to a split G-quadruplex, (ii) binding said split G-quadruplex to a target nucleic acid,

(iii) binding a detectable capture target to said capture tag, and (iv) detecting the detectable capture target with a method known in the art. In another embodiment, target nucleic acids are detected using capture tags and capture targets by (i) associating a capture tag to a split G-quadruplex, (ii) binding a detectable capture target to said capture tag, (iii) binding said split G- quadruplex to a target nucleic acid, and (iv) detecting the detectable capture target with a method known in the art. And in another embodiment, target nucleic acids are detected using capture tags and capture targets by (i) binding a split G-quadruplex to a target nucleic acid, (ii) associating a capture tag to said split G- quadruplex, (iii) binding a detectable capture target to said capture tag, and (iv) detecting the capture target with a method known in the art. In these methods, capture targets that can be detected can be utilized, or capture targets capable of binding detectable molecules can be utilized. Methods of binding capture tags to capture targets, and binding capture targets to detectable molecules, are known in the art. In a preferred embodiment, the target nucleic acid is bound to a solid surface - either (i) before the target nucleic acid is bound to the split G-quadruplex; or (ii) after the target nucleic acid is bound to the split G-quadruplex, but before detection - permitting washing of said target nucleic acid and removal of unbound detectable capture targets or molecules before detection. An illustration of a target nucleic acid associated to a detectable capture target using said methods and a detectable molecule and a solid surface (i.e. preferred embodiment) - is shown in Figure 1 Embodiment 2.

[033] In another embodiment, detection methods can be improved if combined with secondary methods that (i) amplify the signal of detection tags, detectable capture targets, or detectable molecules; or (ii) capture additional detection tags, detectable capture targets, or detectable molecules. For example, methods to amplify signals include methods to improve the stability of detection tags, detectable capture targets, or detectable molecules (ex. addition of dextran to HRP) ; and methods to improve the catalytic activity of detection tags, detectable capture targets, or detectable molecules (ex. addition of PEG to prevent HRP inactivation) . For example, methods to capture (or cascade) additional detection tags, detectable capture targets, or detectable molecules include (i) tyramide signal amplification (TSA) , (ii) avidin-biotinylated enzyme complexes (ABC), and (iii) branched-DNA assays (bDNA) . In one embodiment, the capture arms of a split G-quadruplex are used to capture (or cascade) additional detection tags, detectable capture targets, or detectable molecules - for example, by binding capture nucleic acids that (i) are associated with detection tags or detectable capture tags, or (ii) are capable of binding molecules that can bind or cascade with detectable molecules. In another embodiment, the capture arms of a split G-quadruplex are used to capture (or cascade) additional split G- quadruplexes , that optionally have detection tags or detectable capture tags (ex. additional capture arms capable of binding additional capture nucleic acids or split G-quadruplexes) .

Methods to Capture Nucleic Acids

[034] Methods of using capture tags to bind capture targets are known in the art, and are used to bind nucleic acids - associated with capture tags - to capture targets, including solid surfaces. Nucleic acids bound to solid surfaces are used in several methodologies, including nucleic acid precipitation, nucleic acid purification, branched DNA assays, solid phase PCR amplification, and solid phase bridge amplification (for NGS sequencing) . These methodologies can be grouped into two capture methods, where the first group of capture methods - nucleic acid precipitation and nucleic acid purification - generally use a target nucleic acid associated to a capture tag, which is capable of binding a solid surface; and the second group of capture methods - branched DNA, solid phase PCR, and solid phase bridge amplification - generally use a non-target nucleic acid (which is capable of hybridizing to the target nucleic acid, ex. a primer), which is associated to a capture tag, and thus capable of binding a solid surface. In one embodiment of the present invention, split G-quadruplexes associated to capture tags, that are capable of binding both target nucleic acid (via target-binding arms) and a solid surface (via a capture tag) , can be used to bind target nucleic acids to solid surfaces. Accordingly, in the aforementioned methodologies using nucleic acids bound to solid surfaces, a split G-quadruplex associated to a capture tag plus a nucleic acid can substitute the target nucleic acid associated to a capture tag (in the first group) and the non-target nucleic acid associated to a capture tag plus a nucleic acid (in the second group) of the aforementioned methodologies.

[035] In one embodiment, a split G-quadruplex associated to a capture tag can be used to bind a target nucleic acid to a solid surface by (i) associating a capture tag to the first strand of a split G-quadruplex,

(ii) binding said first strand and the second strand of the split G-quadruplex to a target nucleic acid, and

(iii) bind said capture tag to a solid surface. In a second embodiment, a split G-quadruplex associated to a capture tag can be used to bind a target nucleic acid to a solid surface by (i) associating a capture tag to the first strand of a split G-quadruplex, (ii) binding said capture tag to a solid surface, and (iii) binding the second strand of the split G-quadruplex and the target nucleic acid to the surface bound first strand of the split G-quadruplex. An illustration of a target nucleic acid associated to a capture tag and solid surface using said methods - is shown in Figure 1 Embodiment 4. The illustration also shows an optional detection tag (D) on the second strand of the split G-quadruplex, which permits detection of the captured target nucleic acid, preferentially using an added wash step before the detection step to remove unbound detection tags. In another embodiment, a split G-quadruplex associated to a capture tag can be used to bind target nucleic acids to a solid surface, by (i) associating capture tags to the first and second strands of a split G-quadruplex, (ii) binding said first and second strands of the split G- quadruplex to a target nucleic acid, and (iii) binding said capture tag to a solid surface.

[036] Embodiments for binding nucleic acids to solid surfaces are useful for the first group of capture methodologies (including nucleic acid precipitation and purification) , and are useful for the second group of capture methodologies (including solid phase PCR and bridge amplification) when combined with template- directed nucleic acid synthesis - such as enzymatic methods for DNA synthesis, DNA amplification, DNA transcription, and RNA synthesis. Methods of template- directed nucleic acid synthesis are known in the art, and can be classified in three groups, requiring (i) a primer (or 3' -OH terminus) for initiation, (ii) a promoter sequence for initiation, or (iii) neither primer or promoter for initiation. These requirements for primers or promoters can be accommodated by the split G- quadruplex - with or without association to a capture tag - for example, by using the 3' -OH terminus of the second strand for initiation, or incorporating a promoter sequence (for example, flanking the target binding arm) .

[037] In one embodiment, a method of template- directed nucleic acid synthesis requiring a primer is performed by (i) binding a target nucleic acid to a split G-quadruplex associated to a capture tag, and binding the capture tag to a solid surface; and (ii) initiating nucleic acid synthesis using the 3' -OH terminus of the second strand of the split G-quadruplex. In one preferred embodiment, the 3' -OH terminus of the first strand of the split G-quadruplex is modified (ex. aminated) to prevent synthesis from the first strand. In a second preferred embodiment, the temperatures for target nucleic acid hybridization and enzymatic extension are below the melting temperature of the G-quadruplex. In a second embodiment, a method of template-directed nucleic acid synthesis requiring a promoter is performed by (i) binding a target nucleic acid to a split G-quadruplex associated to a capture tag and a promoter sequence, and binding the capture tag to a solid surface; and (ii) initiating nucleic acid synthesis using the promoter sequence associated to the split G-quadruplex. In a preferred embodiment, the promoter sequence is incorporated in a region flanking the target-binding arm. In another preferred embodiment, the promoter sequence is double-stranded nucleic acid, for example, formed by hybridizing a complementary sequence incorporated in the same or a different strand of the split G-quadruplex, or by hybridizing a complementary sequence incorporated in a nucleic acid fragment.

[038] Template-directed nucleic acid synthesis of the target nucleic acid can be used to strengthen the binding between the split G-quadruplex and the target nucleic acid. For example, if the nucleic acid synthesis is initiated at the 3' -OH terminus of the split G-quadruplex and is extended along the length of the target nucleic acid (downstream of the split G-quadruplex) , it creates a complementary nucleic acid that is connected to the split G-quadruplex and bound to the target nucleic acid. Depending on the length of the target nucleic acid that is downstream of the split G-quadruplex, the newly synthesized complementary fragment can be large, and can be used to strengthen the binding the split G-quadruplex and the target nucleic acid. In one embodiment, the binding of a split G-quadruplex bound to a target nucleic acid is strengthen by (i) binding the split G-quadruplex to a target nucleic acid and (ii) performing template- directed nucleic acid synthesis on the target nucleic acid. In another embodiment, a functional tag can be associated to a split G-quadruplex by (i) binding the split G-quadruplex to a target nucleic acid, and (ii) performing template-directed nucleic acid synthesis on the target nucleic acid utilizing nucleotides bound to functional tags.

[039] Use of split G-quadruplexes for template- directed nucleic acid synthesis has two key advantages over single-stranded primers (with sequence equal to the target-binding arms) : (i) split G-quadruplexes can be designed to hybridize at much different temperatures in comparision to single stranded primers, for example by decreasing the length of one arm and increasing the length of the other arm, while maintaining the same overall sequence; and (ii) split G-quadruplexes are more specific than single-stranded primers because the individual target binding arms can be shorter than primers, and thus more sensitive to nucleotide substitutions, especially if one arm is made short in length .

[040] Examples of commonly used solid surfaces for binding of nucleic acids include glass slides, silicon chips, micro-beads, micro-spheres, and sedimentable and ferromagnetic substances, such as agarose resin and iron beads. Examples of tags (and corresponding reactive groups or coatings on solid surfaces) include amino modifications (and epoxy silane or isothiocynanate coated surfaces), thiol modifications (and mercaptosilanized surfaces), hydrazide modifications (and aldehyde or epoxide) , biotin (and immobilized streptavidin) , cholesterol-TEG (and immobilized anti-cholesterol antibodies), and digoxigenin NHS Ester (and immobilized anti-digoxigenin antibodies) . Some tags bind directly to the reactive groups on the solid surface (ex. biotin and streptavidin) , and other tags require a chemical reaction with secondary chemicals for attachment. Examples of micro-spheres include polystyrene micro-spheres, magnetic micro-spheres, and silica micro-spheres.

[041] Split G-quadruplexes are advantageous for the capture of target nucleic acids, including (1) split G- quadruplexes can selectively capture a target nucleic acid based on sequence, with SNP specificity; (2) split G-quadruplexes can be readily used for template-directed nucleic acid synthesis of the target nucleic acid that has been captured, (3) split G-quadruplex can be easily denatured (ex. thermally, chemically) or digested (ex. at a engineered restriction site by a restriction enzyme) to liberate the target nucleic acid that has been captured, and (4) split G-quadruplexes can be simpler to modify than nucleic acids (ex. long synthetic nucleic acids, and especially nucleic acids isolated from biological sources) . Further, the efficiency of capture can be easily monitored, for example, by (i) capturing a target nucleic acid onto a solid surface with a split G- quadruplex associated to a detection tag (or detectable capture tag) on one strand and a capture tag on the other strand (Figure 1 Embodiment 4), (ii) removing (ex. by washing) the unbound strand with the detection tag, and (iii) monitoring the detection tag of the bound second strand by methods known in the art. Alternatively, one can (i) capture a target nucleic acid onto a solid surface with a split G-quadruplex with a detection tag on one stand (or both strands), and (ii) monitor the assembled split G-quadruplexes by monitoring its catalytic activity.

[042] In one embodiment, the capture tag is associated to a strand of the G-quadrupex via a linker or spacer - for example, Phosphoramidite C3, Hexanediol, and 1 ' , 2 ' -Dideoxyribose, and preferentially Triethylene Glycol or Hexa-Ethyleneglycol . In cases where there is poor binding between the capture tag and the solid surface - ex. due to steric hinderance, incompatible surface charge, incompatible surface hydrophobicity/hydrophilicity - the linker of the capture tag can be made longer. For example, the linker can be made longer adding additional linker molecules to the first linker (ex. (HEG) 5, which is 5 Hexa-Ethyleneglycol linkers) . Or alternatively, the linker can be made longer by adding additional nucleotides (ex. dTio, which is 10 deoxythymines ) between the capture tag and the other parts of the split G-quadruplex (ex. the target binding arm and the partial G-quadruplex sequence) .

[043] In the present invention, split G-quadruplexes and capture nucleic acids can be used together for the capture of target nucleic acids to solid surfaces. In a preferred embodiment, the split G-quadruplex has capture arms capable of binding the capture nucleic acid, and the capture nucleic acid has a capture tag capable of binding the solid surface. In one embodiment, a split G- quadruplex associated to a capture tag can be used to bind a target nucleic acid to a solid surface by (i) associating a capture tag to a capture nucleic acid, (ii) binding said capture tag to a solid surface, (iii) binding the capture arm(s) of a split G-quadrupex to said capture nucleic acid, and (iv) binding a target nucleic acid to the target binding arms of said split G- quadruplex. In a second embodiment, a split G-quadruplex associated to a capture tag can be used to bind a target nucleic acid to a solid surface by (i) associating a capture tag to a capture nucleic acid, (ii) binding said capture tag to a solid surface, (iii) binding the target binding arms of a split G-quadruplex to a target nucleic acid, and (iv) binding the capture arms of said split G- quadruplex to said capture nucleic acid. An illustration of a target nucleic acid associated to a capture tag - i.e. the capture arms of a split G-quadruplex - and a solid surface using said methods, is shown in Figure 1 Embodiment 5. Said target nucleic acid can be detected using a second G-quadruplex associated with a detection tag (or a capture tag capable of binding detectable capture targets) - as shown in Figure 1 Embodiment 6.

[044] The present invention features a kit and an apparatus for using split G-quadruplexes with functional tags, for example, to detect or capture or target or crosslink target nucleic acids. The kit or apparatus can be point-of-care (POC) . In one embodiment, the kit or apparatus includes a split G-quadruplex associated with a functional tag. In another embodiment, the kit or apparatus includes a split G-quadruplex associated with a capture tag, and a solid surface (capable of binding said capture tag) . And in another embodiment, the kit or apparatus includes a split G-quadruplex associated with a capture tag (capable of binding a capture nucleic acid) , a capture nucleic acid with a capture tag (capable of binding a solid surface), and a solid surface.

EXAMPLE S

[045] Other features and advantages of the invention will be apparent from the following examples of the embodiments and from the claims. Example 1

Strong Binding by Split G-Quadruplexes

[046] To observe the binding of split G-quadruplexes on target nucleic acids (in absence of G-quadruplex catalytic activity) , a target nucleic acid with a capture tag was bound to a solid surface, washed, bound with a split G-quadruplex, washed repeatedly, and then the bound split G-quadruplex was detected. The utilized target nucleic acid had a capture tag and sequence 5' -/5Biosg/NN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NNN NAA CCA TTT GGG TGT CCT GAT-3 ' (SEQ ID NO: 1) . The utilized split G-quadruplex, specific for said target nucleic acid, comprised two oligonucleotide strands, with the first strand of sequence 5' -AT CAG GAC AC/iSp9/GGG TTG GG-3' (SEQ ID NO: 2) and the second strand of sequence 5'-GGG TAG GG/iSp9/CCA AAT GG-3' (SEQ ID NO: 3) . Oligonucleotides were custom-made by IDT (Coralville, Iowa) . Other reagents, unless otherwise indicated, were purchased from Sigma-Aldrich (St. Louis, MO) .

[047] Target nucleic acid (100pm) was first bound to a solid surface - Pierce Streptavidin Coated High Capacity Plates (Thermo Fischer Scientific, Waltham, MA) - for 1 hour at 37 °C in Binding Buffer (50mM HEPES (pH 7.4), 20mM KC1, 50mM NaCl, 0.02% Triton X-100 (0.02% v/v) , 50mM MgCl₂) . The plates were washed in Wash Buffer

(TBS, 0.1% BSA, 0.05% Tween-20), and then bound with 100pm split G-quadruplex for 30 minutes at 25^°C in Binding Buffer. Plates were either (i) washed one time or

(ii) washed one time, rested for 30 minutes, and washed again one time. To observe binding, the catalytic activity of the bound split G-quadruplex was triggered in Binding Buffer supplemented with 125mM hemin, lmM H₂O₂, lmM ABTS; and the 420nm absorbance of the resulting product was measured on a SpectraMax Plus 384 spectrophotometer (Molecular Devices, Sunnyvale, CA) . The results are presented in Table 1, and demonstrate that split G-quadruplexes bind target nucleic acids in absence of catalytic activity - and the binding is stable after one wash or two washes with an added rest step.

TABLE 1

Effect of Washes on Binding of Split G-Quadruplex to

Target Nucleic Acids

Cooperative Binding by Split G-Quadruplexes

[048] The strong binding suggests that the two strands of the split G-quadruplex (on binding the target nucleic acid) bind one another via formation of the G- quadruplex, and this binding further stabilizes each strand on the target nucleic acid. To determine if the binding of the two strands of the split G-quadruplex on the target is cooperative, a target nucleic acid with a capture tag (SEQ ID NO: 1) (100pm) was bound to a solid surface for 30 minutes at 25 ^°C, washed, and then bound for 30 minutes at 25^°C with either (i) 100pm of the first strand of a split G-quadruplex (SEQ ID NO: 2) (herein called well 1) or (ii) 100pm of the second strand of a split G-quadruplex (SEQ ID NO: 3) (herein called well 2) or (iii) both strands of a split G-quadruplex (SEQ ID NO: 2 and SEQ ID NO: 3) (herein called well 3) . Afterwards, the supernatant of well 1 (containing unbound SEQ ID NO:

2) and well 2 (containing unbound SEQ ID NO: 3) was transferred to well A containing 100pm of target nucleic acid of sequence 5'-AA CCA TTT GGG TGT CCT GAT-3 ' (SEQ ID NO: 4); and the supernatant of well 3 (containing unbound SEQ ID NO: 2 and SEQ ID NO : 3 ) was transferred to well b containing 100 pm of target nucleic acid of sequence SED ID NO: 4. To observe binding, the catalytic activity of unbound split G-quadruplexes was triggered, and the 420nm absorbance of the resulting product was measured on a spectrophotometer. The results are presented in Table 2 and indicate cooperative binding - that is, more strands are retained on target nucleic acids when the strands are bound jointly (because of cooperative binding) (ex. well

3) and not separately (ex. well 1 and well 2) .

TABLE 2

More Retention of Split G-Quadruplex Strands Bound

Jointly Than Separately

Example 2

Detection of Target Nucleic Acid by Split G-Quadruplex with Capture Tag

[049] The strong binding and the cooperative binding observed in Example 1 - in absence of G-quadruplex catalytic activity - indicates that split G-quadruplexes stably bind target nucleic acids, and accordingly, they can also be used to associate other molecules such as functional tags (bound or incorporated into the split G- quadruplex) to target nucleic acids. To demonstrate that a capture tag of a split G-quadruplex can be associated to a target nucleic acid - and moreover, a detectable capture target can be bound to the capture tag for detection of the target nucleic acid - a target nucleic acid with a capture tag (SEQ ID NO: 1) was bound to a solid surface, washed, bound with a split G-quadruplex with a DIG capture tag (SEQ ID NO: 2 and sequence 5'-GGG TAG GG/iSp9/ CCA AAT GG/3DiG_N/-3' (SEQ ID NO: 5)), washed, bound for 30 minutes at 25^°C with rabbit anti-DIG antibody (Thermo Fischer Scientific) , washed, bound for 30 minutes at 25^°C with anti-rabbit antibody conjugated to HRP (Sigma-Aldrich) , washed, and then the catalytic activity of the HRP was triggered in Binding Buffer supplemented with lmM H₂0₂ and lmM ABTS, and the 420nm absorbance of the resulting product was measured on a spectrophotometer. (The method closely resembles Figure 1 Embodiment 2, except it uses two antibodies instead of one for detection) . Other solutions and methods are the same as Example 1 unless otherwise indicated. The results are presented in Table 3, and demonstrate the use of split G-quadruplexes to (i) associate functional tags, and in particular capture tags, to target nucleic acids, (ii) associate capture targets to target nucleic acids, and (iii) associate detectable molecules to target nucleic acids via binding to split G-quadruplex capture tags . TABLE 3

Detection of Target Nucleic Acid using Split G-Quadruplex with Capture Tag

Example 3

Detection of Target Nucleic Acid by Split G-Quadruplex with Capture Arms

[050] Split G-quadruplexes can be designed with additional target binding arms (herein called capture arms), which are capable of binding additional target nucleic acids (herein called capture nucleic acids) . In the following example, a split G-quadruplex with two target arms and two capture arms was designed [5' -AT CAG GAC AC /iSp9/ GGG TTG GG /iSp9/ ATT AAG TGT-3 ' (SEQ ID NO: 6) and 5'-GGC CAG TTT CAT TTG AGC / iSp9 / GGG TAG GG /iSp9/ CCA AAT GG-3' (SEQ ID NO: 7)], which was capable of binding two capture nucleic acids, that is, two strands of a second split G-quadruplex of sequence [5'- ACA CTT AAT /iSp9/ GGG TTG GG-3' (SEQ ID NO: 8) and 5'- GGG TAG GG /iSp9/ GCT CAA ATG AAA CTG CCC-3' (SEQ ID NO: 9) ] . To demonstrate the methodology, a target nucleic acid with a capture tag (SEQ ID NO: 1) was bound to a solid surface, washed, bound with a split G-quadruplex with two target arms and two capture arms (SEQ ID NO: 6 and SEQ ID NO: 7), washed, and bound with two capture nucleic acids that were also strands of a second split G- quadruplex (SEQ ID NO: 8 and SEQ ID NO: 9), which is capable of assembling into a functional G-quadruplex (see Figure 1 Embodiment 2) .

TABLE 4

Detection of a Capture Nucleic Acid - a 2^nd Split G

Quadruplex

[051] To observe binding of the captured nucleic acids, the catalytic activity of both split G- quadruplexes was triggered, and the 420nm absorbance of the resulting product was measured on a spectrophotometer. Other solutions and methods are the same as Example 1 unless otherwise indicated. The results are presented in Table 4 above, and demonstrate the use of capture arms on split G-quadruplexes to capture other nucleic acids (that is, capture nucleic acids), and the use of said capture nucleic acids for the detection of target nucleic acids. This approach can be used with other types of detectable nucleic acids in place of the second G-quadrupex, for example, nucleic acids with detection tags, and nucleic acids with capture tags capable of binding detectable molecules. The approach can also be used with capture nucleic acids associated with other types of functional tags (ex. capture tags, target tags, crosslinked tags, etc.), which can be used for the capture, targeting, or crosslinking of target nucleic acids . Example 4

Capture of Target Nucleic Acid by Split G-Quadruplex with

Capture Tag

[052] Several types of capture tags are capable of binding solid surfaces. These capture tags, associated to split G-quadruplexes , can be used to bind split G- quadruplexes to solid surfaces. Moreover, these split G- quadruplexes , capable of binding target nucleic acids, can be used to bind (capture) target nucleic acids to solid surfaces. To demonstrate that split G-quadrplexes can be used to capture target nucleic acids onto solid surfaces, one strand of a split G-quadruplex with a biotin capture tag [ 5' -/ 5BiosG/AT CAG GAC AC/iSp9/ GGG TTG GG-3' (SEQ ID NO: 10) ] was bound to a solid surface, washed, bound with a target nucleic acid (SEQ ID NO: 4) and the second strand of the G-quadruplex with a DIG capture tag (SEQ ID NO: 5), washed, bound with rabbit anti-DIG antibody, washed, bound with anti-rabbit antibody conjugated to HRP, washed, and then the catalytic activity of the HRP was triggered in Binding Buffer supplemented with lmM H₂0₂ and lmM ABTS, and the 420nm absorbance of the resulting product was measured on a spectrophotometer. The method is similar to Figure 1 Embodiment 4, with the drawn detection tag replaced with a capture tag, which capable of binding an antibody

(which is capable of binding a 2^nd antibody) . Other solutions and methods are the same as Example 2 unless otherwise indicated. The results are presented in Table 5, and demonstrate the use of split G-quadruplexes to capture target nucleic acids onto solid surfaces. TABLE 5

Capture of a Target Nucleic Acid by Split G Quadruplex with Capture Tag

Example 5

Capture of Assembled Target Nucleic Acid + Split G-

Quadruplex

[053] A second approach to capture target nucleic acids onto solid surfaces binds split G-quadruplexes

(with capture tags) to target nucleic acids, and then binds the (capture tag with the) assembled complex to a solid surface. This approach can be optimized by using a smaller amount of the split G-quadruplex strand with the capture tag relative to its other strand. To demonstrate this approach, a split G-quadruplex with Biotin and DIG capture tags (25pm of SEQ ID NO: 10 and 100pm of SEQ ID NO: 5) was mixed for 60 minutes with 100pm of target nucleic acid, and then bound to a solid surface (coated with avidin) . The bound, assembled complexes were then washed, bound with rabbit anti-DIG antibody, washed, bound with anti-rabbit antibody conjugated to HRP, washed, and then the catalytic activity of the HRP was triggered in Binding Buffer supplemented with lmM H₂O₂ and lmM ABTS, and the 420nm absorbance of the resulting product was measured on a spectrophotometer. Other solutions and methods are the same as Example 4 unless otherwise indicated. The results are presented in Table 6, and demonstrate a second approach to capture target nucleic acids onto solid surfaces using split G- quadruplexes .

TABLE 6

Capture of Assembled Target Nucleic Acid/Split G

Quadruplex with Capture Tag

Claims

WHAT IS CLAIMED IS:

1. A method of associating a tag to a nucleic acid, comprising: associating the tag to a split G-quadruplex, and binding the split G-quadruplex to the nucleic acid.

2. The method of claim 1, further comprising performing template-directed nucleic acid synthesis of the nucleic acid .

3. The method of claim 1, wherein the associating step is accomplished by binding or incorporating the tag into one strand of the split G-quadruplex.

4. The method of claim 1, wherein the associating step is accomplished by binding or incorporating the tag into two strands of the split G-quadruplex.

5. The method of claim 1, wherein the tag is an attachment chemistry, a fluorophore, a detectable enzyme, a detectable particle, or a nucleotide analog.

6. The method of claim 1, wherein the tag is a detection tag, a capture tag, a targeting tag, or a crosslinking tag.

7. The method of claim 6, wherein the capture tag is a capture arm.

8. The method of claim 1, wherein the tag is associated to the split G-quadruplex by template-directed nucleic acid synthesis.

9. A method of detecting a nucleic acid, comprising: associating a detection tag or capture tag to a split G- quadruplex, binding the split G-quadruplex to the nucleic acid, and detecting the detection tag or capture tag.

10. The method of claim 9, wherein the capture tag is detected by binding a detectable capture target to the capture tag, and detecting the detectable capture target.

11. The method of claim 9, further comprising binding the nucleic acid to a solid surface.

12. The method of claim 11, comprising: associating a detection tag or capture tag to a split G-quadruplex, binding the capture tag if present to a detectable capture target, binding the split G-quadruplex to a nucleic acid, binding the nucleic acid to a solid surface, washing the solid surface to remove unbound molecules, and detecting the bound detection tag or detectable capture target.

13. The method of claim 9, wherein the detection tag or capture tag is associated to the split G-quadruplex by template-directed nucleic acid synthesis.

14. A method of capturing a nucleic acid, comprising: associating a capture tag to a split G-quadruplex, binding the split G-quadruplex to the nucleic acid, and binding the capture tag to a capture target.

15. The method of claim 14, wherein the capture target is a nucleic acid, a molecule that can be detected, a molecule that can bind other molecules, or a solid surface .

16. The method of claim 15, wherein the capture target is a solid surface.

17. The method of claim 15, wherein the capture tag is a capture arm, and the capture target is a capture nucleic acid .

18. The method of claim 17, further comprising binding the capture nucleic acid to a solid surface.

19. The method of claim 14, wherein the split G- quadruplex has a detection tag or a second capture tag capable of binding a detectable capture target.

20. The method of claim 14, wherein the capture tag is associated to the split G-quadruplex by template-directed nucleic acid synthesis.