Binding molecules built from L-DNA with added nucleotides

Abstract

This invention provides for processes for creating a long DNA molecule that is a concatamer that comprises a repeating oligonucleotide segment, wherein one or more of the nucleotides of said segment has one or more independently selected templating “non-standard” nucleotides. These are nucleotide analogs that, when incorporated into oligonucleotides (DNA or RNA, collectively xNA), present to a pattern of hydrogen bonds that is different from the pattern presented by adenine, guanine, cytosine, and uracil. This disclosure provides a process for obtaining these using rolling circle amplification.

Claims

1. A process to create an oligonucleotide concatamer, said concatamer comprising a repeating oligonucleotide segment, wherein at least one of the nucleotides of said segment has a templating nucleobase selected from the group consisting of ##STR00001## wherein R is the point of attachment of said templating nucleobase to said oligonucleotide segment, wherein said process comprises contacting a circular oligonucleotide that comprises a segment that is Watson-Crick complementary to said repeating oligonucleotide segment with (i) a primer, (ii) a strand displacing polymerase, and (iii) a group of nucleoside triphosphates, wherein said nucleobases of said group complement all of the nucleobases in said circular oligonucleotide.

2. The process of claim 1, wherein said strand displacing polymerase is the phi 29 DNA polymerase.

3. The process of claim 1, wherein said nucleobase is ##STR00002## where R is the point of attachment of said nucleobase to said oligonucleotide.

4. The process of claim 1, wherein said nucleobase is ##STR00003## where R is the point of attachment of said nucleobase to said oligonucleotide.

5. The process of claim 1, wherein said nucleobase is ##STR00004## where R is the point of attachment of said nucleobase to said oligonucleotide.

6. The process of claim 1, wherein said nucleobase is ##STR00005## where R is the point of attachment of said nucleobase to said oligonucleotide.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. Watson-Crick pairing rules follow two rules of complementarity: (a) size complementarity (large purines pair with small pyrimidines) and (b) hydrogen bonding complementarity (hydrogen bond donors, D, pair with hydrogen bond acceptors A). Rearranging D and A groups on various heterocycles supports an artificially expanded genetic information system (AEGIS). These are the presently most preferred heterocycles for the instant invention. The nucleotides herein that are not found in natural DNA or RNA are called “non-standard”.

(2) FIG. 2. This invention teaches the different heterocycles can implement the same hydrogen bonding pattern. Shown here are presently preferred heterocycles for implementing the instant invention, as alternatives. The nucleotides herein that are not found in natural DNA or RNA are called “non-standard”.

(3) FIG. 3. Synthetic route generates a glycal having the L-configuration. This is a precursor for synthesizing all C-glycosides in the instant invention, specifically from FIG. 1, the heterocycles implementing the V, K, and Z hydrogen bonding patterns, and in FIG. 2, the heterocycles implementing the S, V, K, and Z hydrogen bonding patterns following routes covered in the parents of the instant application. This drawing also described the route to prepare the FIG. 1 AEGIS S in its epimeric form.

(4) FIG. 4. Synthetic route to a protected phosphoramidite of AEGIS Z follows routes that are described in the parents. The precursor is commercially available from Carbosynth.

(5) FIG. 5. Synthetic route to generate L-AEGIS P. Other AEGIS N-glycoside analogs are prepared analogously following routes described for the D-enantiomer in the parents.

(6) FIG. 6. Rolling circle amplification (RCA) is disclosing this diagram as an alternative to PCR way to amplify survivors from an AEGIS-LIVE, the process disclosed in the parents. Here, instead of the primer binding sites (PBS) being used to bind primers in the PCR, those same sites are used to anneal to a splint which allows the ligation of the survivors to form a circle. The splint can then be used as a primer for rolling circle amplification, leading to between 10,000 and 100,000 copies of the survivor sequence(s) as a linear concatamer. These can then be cleaved by a restriction enzyme placed strategically to yield the same number of short survivor sequence(s) which can be introduced into AEGIS-LIVE. The presently preferred polymerase for the RCA is the Phi 29 polymerase, which is commercially available, Bst I or other strand displacing polymerase.

(7) FIG. 7. Polyacrylamide gel electrophoresis (PAGE) analysis of results of treating a library of oligonucleotides built from a six letter genetic alphabet, with a random region of 30 nucleotides embedded within two primer sections as shown in SEQ ID 1, with Circligase™ (Lucigen). The circuligation is indicated by a band running similar to a single-stranded oligonucleotide 120 nucleotides long (Lane 2). The uncircularized starting material can be removed by exonuclease 1 (Lane 3). The linear material is shown in Lanes 4 and 5. The sequence of the library is: 5′ CTA GCA ACC AGC ACG AAG C (N).sub.30 ACA GGA GCA GGT CGT GT3′, which is equivalent to:

(8) TABLE-US-00001 SEQ ID 1 CTAGCAACCAGCACGAAGC NNNNNNNNNN NNNNNNNNNN  NNNNNNNNNN ACAGGAGCAGGTCGTGT

(9) FIG. 8. PAGE analysis of the results of amplification of the circularized product obtained using Circligase™ in a Phi 29/Phi 29 polymerase/buffer system including either a standard (A/C/G/T) or AEGIS containing (A/C/G/T/Z/P) dNTP mix using polyacrylamide gel electrophoresis (PAGE) with α-[.sup.32P] GTP. With just four standard triphosphates, product (the broad band between 300 and 1800 nts) is not seen. RCA products are sonicated before PAGE. ACGTZP N30 library (CN30).

DESCRIPTION OF INVENTION

(10) Definition of Non-Standard Components of an Artificially Expanded Genetic Information System

(11) This application teaches a distinction between the hydrogen-bonding pattern (in FIG. 1 and FIG. 2 nomenclature, pyDAD, for example) and the heterocycle that implements it. Thus, the pyADA hydrogen-bonding pattern is implemented by thymidine, uridine, and pseudouridine. The puDDA hydrogen bonding pattern is implemented by both the heterocycle isoguanosine and 7-deaz-isoguanosine. Heterocycles to implement any particular pre-selected hydrogen-bonding pattern are preferred depending on their chemical properties, for example, high chemical stability or low tautomeric ambiguity. The pyADA, pyDAA, puADD, and puDAD hydrogen bonding patterns are said to be “standard” hydrogen bonding patterns, and to form with their appropriate complement “standard base pairs”. Other hydrogen bonding patterns are said to be “non-standard”, and to form with their appropriate complement “non-standard base pairs”.

(12) Creating AEGIS-Containing Oligonucleotides Having all of the Carbohydrate Suitors in the L-Configuration

(13) The strategy to generate binding and reactive AEGIS sequences that are stable in cancer-relevant biological environments relies in our ability to present them in their mirror-image form, These in AEGIS-free oligonucleotides are not be substrates for any natural nucleases, including those found in human blood and tissues; mirror image xNA is stable in blood, for example, for as long as 72 hours [Kim, K. R., Lee, T., Kim, B. S., & Ahn, D. R. (2014). Utilizing the bioorthogonal base-pairing system of L-DNA to design ideal DNA nanocarriers for enhanced delivery of nucleic acid cargos. Chem. Sci. 5, 1533-1537.].

(14) Synthesis of L-AEGIS oligonucleotides is implemented using solid phase phosphoramidite chemistry, well known in the art. The only difference is that the phosphoramidite building blocks have the L configuration, and are prepared by one of the processes described in the drawings.

(15) The sequence for the L-AEGIS oligonucleotide(s) that bind and/or chemically transform to an achiral target is obtained simply by following the processes disclosed in the parents. Since the target is not chiral, the L-AEGIS oligonucleotide will bind to and/or chemically transform that achiral target with exactly the same affinity and/or exactly the same rate as the D-AEGIS oligonucleotide.

(16) The sequence for the L-AEGIS oligonucleotide(s) that bind and/or chemically transform to a chiral target is obtained by following the processes disclosed in the parents, except by using the target in its mirror image enantiomeric form. The processes disclosed in the parents will generate D-AEGIS oligonucleotide(s) that bind to and/or chemically transform that chiral target in the form that is the mirror image of the desired target, Then, by symmetry laws in physics, the L-AEGIS oligonucleotide will bind to and/or chemically transform the chiral target in the desired enantiomeric form with exactly the same affinity and/or exactly the same rate as the D-AEGIS oligonucleotide binds to and/or transforms the target in its mirror image enantiomeric form.

(17) When the desired target is a natural translated protein, which is built from L-amino acids, the target must be the same protein sequence, except built from D-amino acids, Kent and his colleagues have used convergent synthesis to make mirror-image proteins that are arbitrarily large [Kent, S. B. (2009). Total chemical synthesis of proteins. Chemical Society Reviews 38, 338-351]. Alternatively, AEGIS-LIVE may be targeted against a surface loop peptide of a target protein, preferably a flexible surface loop peptide, in the target protein, but synthetic so that it is built from D-amino acids [Rowlands, D. J., Clarke, B. E., Carroll, A. R., Brown, F., Nicholson, B. H., Bittle, J. L., Houghten, R. A. & Lerner, R. A. (1983) Nature (London) 306, 694-697.][Alexander, H., Johnson, D. A., Rosen, J., Jerabek, L., Green, N., Weissman, I. L. & Lerner, R. A. (1983) Nature (London) 306, 697-699.] [Geysen, H. M., Barteling, S. J., & Meloen, R. H. (1985). Small peptides induce antibodies with a sequence and structural requirement for binding antigen comparable to antibodies raised against the native protein. Proc. Natl. Acad. Sci. USA 82, 178-182.]. This presently preferred process follows rules to how to extract peptides from a full protein to serve for this purpose [Walter, G. (1986). Production and use of antibodies against synthetic peptides. J. Immunol. Meth. 88, 149-161.

(18) To force the peptide to adopt a turn conformation, the presently preferred implementation places cysteines at the end of the peptide. These form a cyclic disulfide is conformation resembles that of the natural were turned in a natural protein. Again, the amino acids must have the D-configuration.

(19) The currently claimed method for implementing this involves a rolling circle amplification, or RCA. Here, a circular oligonucleotide is created from linear nucleotide in any of a number of ways. This includes a ligation exploits of splint. It also includes using an enzyme called Circligase™, which does not require a splint. The presently preferred circular oligonucleotides are between 50 and 200 nucleotides in length. They embody a segment that contains one or more nonstandard nucleotides, specifically preferred or those shown in FIG. 1. The most preferred of those in that figure are designated P and Z in the literature.

(20) Once circularized, a strand displacing DNA polymerase can extend a primer that is annealed to the circle. This requires the appropriate buffer, temperature, and salt concentrations, as well as nucleoside triphosphates. This primer extension generates what is known as a concatamer. This concatamer is a long oligonucleotide that contains repeating segments that are complementary to the sequence of the DNA circle that is acting as a template. For successful concatamer formation, all the triphosphates must be present, that is, each and every nucleotide in the templating DNA circle must find in the solution the triphosphate of the complementary nucleoside. For example, if the circularized DNA template contains a dP, then the RCA requires the mixture to contain the complementary dZTP. Likewise, if the circularized DNA template contains a dZ, then the RCA requires the mixture to contain the complementary dPTP. Presently preferred are templates that contain dZ and dP, meaning that the presently preferred triphosphate mixtures contain dPTP and dZTP. Also likewise, the standard triphosphates must be present in the RCA incubation mixture as necessary to complement the standard templating nucleotides in the circular template.

EXAMPLES

Example 1. Synthesis of a Glycal Having the Unnatural L-Configuration

(21) The mirror image L-nucleotides for G, A, T, and C needed to construct the L-AEGISbodies are commercially available. However, the L-AEGIS nucleotides that are used to synthesize the L-AEGISbodies are not. The precursor for the AEGIS components that are C-glycosides is prepared by the literature route shown in FIG. 3. Arabinose, a very inexpensive L-sugar, is the precursor. The same route, adapted from the parents for the L-glycal, leads to the formation of RNA AEGIS building blocks.

Example 2. Synthesis of a L-AEGIS Nucleoside Phosphoramidites Suitable for Solid Phase DNA Synthesis, Here Implementing the Z Hydrogen Bonding Pattern from FIG. 2

(22) An alternative route to C-glycosidic AEGIS components that are C-glycosides is shown in FIG. 4. This alternative route starts from L-thymidine, which is presently available from Carbosynth for $750/25 grams. This also follows synthetic procedures disclose in the parents. The inversion of configuration does not change any of the chemistry involved.

Example 3. Synthesis of a L-AEGIS Nucleoside Phosphoramidites Suited for Solid Phase DNA Synthesis, Implementing the P Hydrogen Bonding Pattern from FIG. 1 and FIG. 2

(23) The route to prepare N-glycosidic AEGIS components in a protected form. This starts with the-L chlorosugar. Again, this follows synthetic procedures disclose in the parents. The inversion of configuration does not change any of the chemistry involved.

Example 4. Preparing the Circularized Library

(24) As unpredictable arts, chemistry and biochemistry require experimental validation to establish without undue experimentation that an enzyme that can be used to create circular libraries of standard DNA nucleotides actually works when the oligonucleotides being circularized contain nonstandard nucleotides, and that enzymes capable of doing rolling circle amplification (RCA) with standard nucleotides can indeed copy DNA containing nonstandard nucleotides.

(25) In this example, we discover unexpectedly that this is the case with both dP and dZ (FIG. 1). This example compares RCA amplification of a circularized “ACGTZP N30” library using the strand displacing Phi 29 DNA polymerase in aPhi 29 DNA polymerase reaction buffer system.

(26) Here, SEQ ID 1 (Molar Extinction Coefficient: 1.63×10.sup.−7) was treated with Circligase™ (Lucigen) under conditions recommended by the manufacturer. The products were then analyzed by PAGE. Successful circuligation is indicated by a band running similar to a single-stranded oligonucleotide 120 nucleotides long (Lane 2, FIG. 7). The unciruclized starting material can be removed by exonuclease 1 (Lane 3). The linear material is shown in Lanes 4 and 5.

(27) This material was then contacted with a primer in a RCA reaction mixture:

(28) 5′-GACACGACCTGCTCCTGT-3′ SEQ ID 2

(29) RCA reaction mixtures (20 μL) contained the following: 0.07 μM purified circularized ACGTZP N30 Library (CN30) (from SEQ ID 1), 1 μM of primer (SEQ ID 2), 1× of Phi 29 reaction buffer (50 mM Tris HCL, 10 mM (NH.sub.4).sub.2SO.sub.4, 10 mM MgCl.sub.2, and 4 mM DTT at, pH 7.5), 0.125 μM BSA, dNTP mix (either dA/dC/dT or dA/dC/dT/dZ/dP at 1 mM combined with unlabeled dGTP at 0.7 mM and 0.01 μM .sup.32P alpha dGTP), 0.4 U/μL of the Phi 29 polymerase and water (q.s.).

(30) For each sample, a 10 μL solution of template, primer and 1× reaction buffer is heated to 85° C. for 1 minute and cooled to R°T to anneal primer to template.

(31) Ten microliters of a reaction mix containing the Phi 29 polymerase/reaction buffer along with cold dNTPs, .sup.32P alpha dGTP and water (q.s.) were added to each cooled annealed primer/template sample. Samples were incubated for 2 hours at 30° C.

(32) Following incubation the 20 μL RCA reaction mixtures were diluted to 1 to 2 with water (40 μL total). The diluted RCA products were sonicated at room temperature with 3×10 second bursts with a 10 second interval between. Upon sonication, the high molecular weight CN30 RCA product (1800 nt to approximately 200 nt).

(33) The noted feature of the gel shown in FIG. 8 is that RCA product is seen when all six triphosphates were presented (dATP, dCTP, dGTP, TTP, dZTP, dPTP), while no RCA product is detectable when dZTP and dPTP are absent. This feature is quantitated by densitometry measurements of the RCA product (top “smear”) versus the free .sup.32P alpha dGTP (bottom band) as the radiolabel incorporation percentages are 32% versus 84% with standard and the dZTP, dPTP containing dNTP mixes, respectively. These results indicate that the presence of Z and P greatly enhances the amount of RCA product, and, in turn, leads to the conclusion that Phi 29 polymerase is able to incorporate both dZ and dP into a concatamer product under these conditions. It further drives the conclusion that in substantial amounts of dZ and dP are lost through mismatches of template dZ and dP should dZTP and dPTP be absent.