Shuichi Hoshika

In vitro selection with artificial expanded genetic information systems

Significance Many chemicals are valuable because they bind to other molecules. Chemical theory cannot directly design “binders.” However, we might recreate in the laboratory the Darwinian processes that nature uses to create binders. This in vitro evolution uses nucleic acids as binders, libraries of DNA/RNA to survive a selection challenge before they can have “children” (systematic evolution of ligands by exponential enrichment, SELEX). Unfortunately, with only four nucleotides, natural DNA/RNA often yields only poor binders, perhaps because they are built from only four building blocks. Synthetic biology has increased the number of DNA/RNA building blocks, with tools to sequence, PCR amplify, and clone artificially expanded genetic information systems (AEGISs). We report here the first example of a SELEX using AEGIS, producing a molecule that binds to cancer cells. Artificially expanded genetic information systems (AEGISs) are unnatural forms of DNA that increase the number of independently replicating nucleotide building blocks. To do this, AEGIS pairs are joined by different arrangements of hydrogen bond donor and acceptor groups, all while retaining their Watson–Crick geometries. We report here a unique case where AEGIS DNA has been used to execute a systematic evolution of ligands by exponential enrichment (SELEX) experiment. This AEGIS–SELEX was designed to create AEGIS oligonucleotides that bind to a line of breast cancer cells. AEGIS–SELEX delivered an AEGIS aptamer (ZAP-2012) built from six different kinds of nucleotides (the standard G, A, C, and T, and the AEGIS nonstandard P and Z nucleotides, the last having a nitro functionality not found in standard DNA). ZAP-2012 has a dissociation constant of 30 nM against these cells. The affinity is diminished or lost when Z or P (or both) is replaced by standard nucleotides and compares well with affinities of standard GACT aptamers selected against cell lines using standard SELEX. The success of AEGIS–SELEX relies on various innovations, including (i) the ability to synthesize GACTZP libraries, (ii) polymerases that PCR amplify GACTZP DNA with little loss of the AEGIS nonstandard nucleotides, and (iii) technologies to deep sequence GACTZP DNA survivors. These results take the next step toward expanding the power and utility of SELEX and offer an AEGIS–SELEX that could possibly generate receptors, ligands, and catalysts having sequence diversities nearer to that displayed by proteins.

RNA interference induced by siRNAs modified with 4′‐thioribonucleosides in cultured mammalian cells

Short interfering RNAs (siRNAs) variously modified with 4′‐thioribonucleosides against the Photinus luciferase gene were tested for their induction of the RNA interference (RNAi) activity in cultured NIH/3T3 cells. Results indicated that modifications at the sense‐strand were well tolerated for RNAi activity except for full modification with 4′‐thioribonucleosides. However, the activity of siRNAs modified at the antisense‐strand was dependent on the position and the number of modifications with 4′‐thioribonucleosides. Since modifications of siRNAs with 4′‐thioribonucleosides were well tolerated in RNAi activity compared with that of 2′‐O‐methyl nucleosides, 4′‐thioribonucleosides might be potentially useful in the development of novel and effective chemically modified siRNAs.

Study of Modification Pattern–RNAi Activity Relationships by Using siRNAs Modified with 4′-Thioribonucleosides†

A detailed study of the modification pattern–RNAi activity relationships by using siRNAs that are modified with 4′‐thioribonucleosides has been carried out against photinus luciferase and renilla luciferase genes in cultured mammalian NIH/3T3, HeLa, and MIA PaCa‐2 cell lines. When the photinus luciferase gene was targeted, all of the modified siRNAs showed activity equal to, or less than the unmodifed siRNA. In contrast, all modified siRNAs that have a similar modification pattern showed activity equal to or much higher than the unmodified siRNA when tested with the renilla luciferase gene. These results indicated that siRNAs such as RNA33 and RNA53, which each have four residues of the 4′‐thioribonucleoside unit on both ends of the sense strand and four residues on the 3′‐end of the antisense strand, were the most effective. Accordingly, we succeeded in developing modified siRNAs that have the greatest number of 4′‐thioribonucleosides without loss of RNAi activity, and that exhibit potent RNAi activity against two target genes in three different cell lines. Our findings also indicate the significance of target sequences and cell lines when RNAi activity is compared with that of the unmodified siRNA.

Artificial Genetic Systems: Self-Avoiding DNA in PCR and Multiplexed PCR†

Many applications of DNA chemistry in biology and medicine would be enhanced if procedures for the efficient analysis of single DNA molecules also worked well for the analysis of many DNA molecules (multiplexing). Unfortunately, multiplexing often requires the addition of many DNA probes and primers to an assay at the same time, often in great excess with respect to the targeted DNA molecules. Multiple primers built from standard nucleotides can easily interact with each other, even when well-designed. These interactions can create artifacts and noise that defeat the analysis, especially when polymerases are involved in the analytic architecture, as in multiplexed PCR. With more than a dozen target amplicons, multiplexed PCR generally fails because of PCR artifacts. Recently, we reported that the efficiency and consistency of multiplexed PCR could be greatly improved by placing components of our artificially expanded genetic information system (AEGIS) in the external primers in a nested PCR architecture. AEGIS increases the number of independently replicable nucleotides from the natural four (A, T, G, and C) to as many as 12. AEGIS is now in the clinic, where it personalizes the care of some 400 000 patients annually infected with the HIV, hepatitis B, and hepatitis C viruses. However, a nested PCR architecture still does not prevent the analyte-specific segments of the chimeric primers from interacting with each other, as these segments must be constructed from natural nucleotides. In a different strategy, multiplexed PCR might be enabled if the analyte-specific portions of the primers were built from a “self-avoiding molecular-recognition system” (SAMRS). SAMRS DNA can be viewed as the opposite of AEGIS DNA in that it binds to natural DNA, but not to other members of the same SAMRS species. Schematically, an SAMRS replaces T, A, G, and C with the nucleotide analogues T*, A*, G* and C*, whereby T* pairs with A, A* pairs with T, G* pairs with C, and C* pairs with G, but neither the T*–A* pair nor the G*– C* pair contributes substantially to the stability of a duplex. In particular, if PCR primers were built from SAMRS components, they should enable multiplexed PCR without artifacts arising from primer–primer interactions. Empirical studies have shown that pairs joined by two hydrogen bonds contribute to duplex stability, but not pairs joined by one hydrogen bond. Accordingly, a candidate for G* in a “first-generation” SAMRS heterocycle might be hypoxanthine (found in inosine), which pairs with C by using the top two hydrogen-bonding units of C (Scheme 1, top left). The corresponding first-generation candidate for C* would be pyrimidin-2-one (found in zebularine), which pairs with standard G by using the bottom two hydrogen-bonding units of G (Scheme 1, top left). As hypoxanthine and pyrimidin-2one can form only one hydrogen bond in a standard Watson– Crick arrangement, the resulting pair should not contribute to duplex stability; the inosine–zebularine pair would be a G*– C* self-avoiding pair. For the second self-avoiding pair, pyridone might be a first-generation T* candidate. It would pair with standard A by using the top two hydrogen-bonding units of A (Scheme 1, top right). As standard adenine lacks a “bottom” hydrogenbonding unit, 2-aminopurine would be a candidate for A*: it would pair with standard T at the bottom two sites. 2Aminopurine and pyridone would form only one hydrogen bond (Scheme 1, top right) and therefore would not contribute to duplex stability. The aminopurine–pyridone pair would then be an A*–T* self-avoiding pair. Some representative melting temperatures of duplexes incorporating these SAMRS components are shown in Tables 1 and 2 of the Supporting Information. SAMRS should be effective for simple binding assays. For example, in 1996, Kutyavin et al. reported that “pseudocomplementary” diaminopurine and 2-thiothymine bound to thymine and adenine, respectively, but that diaminopurine did not bind to 2-thiothymine. The use of 2-thiothymine instead of pyridone as a T* candidate is consistent with a need for minor-groove solvation to stabilize double helices. Indeed, 2-thiothymine pairs with A slightly better than T itself (see Table 3 in the Supporting Information). Pseudocomplementarity of this limited type has been used in peptide nucleic acids (PNAs) to invade duplex DNA. Gamper and co-workers showed that similar species could be incorporated into DNA as triphosphates, and suggested that the products from this incorporation might not fold and might therefore be more uniformly captured on arrays. 12] Accordingly, we attempted to extend the SAMRS concept to PCR by incorporating various SAMRS candidates into PCR primers on the basis of what we learned by analyzing duplexes built from a first-generation SAMRS alphabet (see Tables 1 and 2 in the Supporting Information). We encountered multiple difficulties. First, 2’-deoxy-5-methylzebularine [*] Dr. S. Hoshika, Dr. F. Chen, Dr. N. A. Leal, Dr. S. A. Benner Foundation for Applied Molecular Evolution The Westheimer Institute for Science and Technology 720 SW 2nd Avenue, Suite 201, Gainesville, FL 32601 (USA) Fax: (+ 1)352-271-7076 E-mail: sbenner@ffame.org Homepage: http://www.ffame.org

Aptamers against Cells Overexpressing Glypican 3 from Expanded Genetic Systems Combined with Cell Engineering and Laboratory Evolution

Laboratory in vitro evolution (LIVE) might deliver DNA aptamers that bind proteins expressed on the surface of cells. In this work, we used cell engineering to place glypican 3 (GPC3), a possible marker for liver cancer theranostics, on the surface of a liver cell line. Libraries were then built from a six-letter genetic alphabet containing the standard nucleobases and two added nucleobases (2-amino-8H-imidazo[1,2-a][1,3,5]triazin-4-one and 6-amino-5-nitropyridin-2-one), Watson-Crick complements from an artificially expanded genetic information system (AEGIS). With counterselection against non-engineered cells, eight AEGIS-containing aptamers were recovered. Five bound selectively to GPC3-overexpressing cells. This selection-counterselection scheme had acceptable statistics, notwithstanding the possibility that cells engineered to overexpress GPC3 might also express different off-target proteins. This is the first example of such a combination.

Laboratory evolution of artificially expanded DNA gives redesignable aptamers that target the toxic form of anthrax protective antigen

Reported here is a laboratory in vitro evolution (LIVE) experiment based on an artificially expanded genetic information system (AEGIS). This experiment delivers the first example of an AEGIS aptamer that binds to an isolated protein target, the first whose structural contact with its target has been outlined and the first to inhibit biologically important activities of its target, the protective antigen from Bacillus anthracis. We show how rational design based on secondary structure predictions can also direct the use of AEGIS to improve the stability and binding of the aptamer to its target. The final aptamer has a dissociation constant of ∼35 nM. These results illustrate the value of AEGIS-LIVE for those seeking to obtain receptors and ligands without the complexities of medicinal chemistry, and also challenge the biophysical community to develop new tools to analyze the spectroscopic signatures of new DNA folds that will emerge in synthetic genetic systems replacing standard DNA and RNA as platforms for LIVE.

Recombinase-based isothermal amplification of nucleic acids with self-avoiding molecular recognition systems (SAMRS).

Recombinase polymerase amplification (RPA) is an isothermal method to amplify nucleic acid sequences without the temperature cycling that classical PCR uses. Instead of using heat to denature the DNA duplex, RPA uses recombination enzymes to swap single‐stranded primers into the duplex DNA product; these are then extended using a strand‐displacing polymerase to complete the cycle. Because RPA runs at low temperatures, it never forces the system to recreate base‐pairs following Watson–Crick rules, and therefore it produces undesired products that impede the amplification of the desired product, complicating downstream analysis. Herein, we show that most of these undesired side products can be avoided if the primers contain components of a self‐avoiding molecular recognition system (SAMRS). Given the precision that is necessary in the recombination systems for them to function biologically, it is surprising that they accept SAMRS. SAMRS‐RPA is expected to be a powerful tool within the range of amplification techniques available to scientists.

Ribonucleosides for an artificially expanded genetic information system.

Rearranging hydrogen bonding groups adds nucleobases to an artificially expanded genetic information system (AEGIS), pairing orthogonally to standard nucleotides. We report here a large-scale synthesis of the AEGIS nucleotide carrying 2-amino-3-nitropyridin-6-one (trivially Z) via Heck coupling and a hydroboration/oxidation sequence. RiboZ is more stable against epimerization than its 2′-deoxyribo analogue. Further, T7 RNA polymerase incorporates ZTP opposite its Watson–Crick complement, imidazo[1,2-a]-1,3,5-triazin-4(8H)one (trivially P), laying grounds for using this “second-generation” AEGIS Z:P pair to add amino acids encoded by mRNA.

Incorporation of Multiple Sequential Pseudothymidines by DNA Polymerases and Their Impact on DNA Duplex Structure

Thermal denaturation and circular dichroism studies suggested that multiple (up to 12), sequential pseudothymidines, a representative C-glycoside, do not perturb the structure of a representative DNA duplex. Further, various Family A and B DNA polymerases were found to extend a primer by incorporating four sequential pseudothymidine triphosphates, and then continue the extension to generate full-length product. Detailed studies showed that Taq polymerase incorporated up to five sequential C-glycosides, but not more. These results constrain architectures for sequencing, quantitating, and analyzing DNA analogs that exploit C-glycosides, and define better the challenge of creating a synthetic biology using these with natural polymerases.

Helicase‐Dependent Isothermal Amplification of DNA and RNA by Using Self‐Avoiding Molecular Recognition Systems

Assays that detect DNA or RNA (xNA) are highly sensitive, as small amounts of xNA can be amplified by PCR. Unfortunately, PCR is inconvenient in low‐resource environments, and requires equipment and power that might not be available in these environments. Isothermal procedures, which avoid thermal cycling, are often confounded by primer dimers, off‐target priming, and other artifacts. Here, we show how a “self avoiding molecular recognition system” (SAMRS) eliminates these artifacts and gives clean amplicons in a helicase‐dependent isothermal amplification (SAMRS‐HDA). We also show that incorporating SAMRS into the 3′‐ends of primers facilitates the design and screening of primers for HDA assays. Finally, we show that SAMRS‐HDA can be twofold multiplexed, difficult to achieve with HDA using standard primers. Thus, SAMRS‐HDA is a more versatile approach than standard HDA, with a broader applicability for xNA‐targeted diagnostics and research.