Nicole A. Leal

Reconstructed evolutionary adaptive paths give polymerases accepting reversible terminators for sequencing and SNP detection

Any system, natural or human-made, is better understood if we analyze both its history and its structure. Here we combine structural analyses with a “Reconstructed Evolutionary Adaptive Path” (REAP) analysis that used the evolutionary and functional history of DNA polymerases to replace amino acids to enable polymerases to accept a new class of triphosphate substrates, those having their 3′-OH ends blocked as a 3′-ONH2 group (dNTP-ONH2). Analogous to widely used 2′,3′-dideoxynucleoside triphosphates (ddNTPs), dNTP-ONH2s terminate primer extension. Unlike ddNTPs, however, primer extension can be resumed by cleaving an O-N bond to restore an -OH group to the 3′-end of the primer. REAP combined with crystallographic analyses identified 35 sites where replacements might improve the ability of Taq to accept dNTP-ONH2s. A library of 93 Taq variants, each having replacements at three or four of these sites, held eight variants having improved ability to accept dNTP-ONH2 substrates. Two of these (A597T, L616A, F667Y, E745H, and E520G, K540I, L616A) performed notably well. The second variant incorporated both dNTP-ONH2sand ddNTPs faithfully and efficiently, supporting extension-cleavage-extension cycles applicable in parallel sequencing and in SNP detection through competition between reversible and irreversible terminators. Dissecting these results showed that one replacement (L616A), not previously identified, allows Taq to incorporate both reversible and irreversible terminators. Modeling showed how L616A might open space behind Phe-667, allowing it to move to accommodate the larger 3′-substituent. This work provides polymerases for DNA analyses and shows how evolutionary analyses help explore relationships between structure and function in proteins.

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

Conversion strategy using an expanded genetic alphabet to assay nucleic acids.

Methods to detect DNA and RNA (collectively xNA) are easily plagued by noise, false positives, and false negatives, especially with increasing levels of multiplexing in complex assay mixtures. Here, we describe assay architectures that mitigate these problems by converting standard xNA analyte sequences into sequences that incorporate nonstandard nucleotides (Z and P). Z and P are extra DNA building blocks that form tight nonstandard base pairs without cross-binding to natural oligonucleotides containing G, A, C, and T (GACT). The resulting improvements are assessed in an assay that inverts the standard Luminex xTAG architecture, placing a biotin on a primer (rather than on a triphosphate). This primer is extended on the target to create a standard GACT extension product that is captured by a CTGA oligonucleotide attached to a Luminex bead. By using conversion, a polymerase incorporates dZTP opposite template dG in the absence of dCTP. This creates a Z-containing extension product that is captured by a bead-bound oligonucleotide containing P, which binds selectively to Z. The assay with conversion produces higher signals than the assay without conversion, possibly because the Z/P pair is stronger than the C/G pair. These architectures improve the ability of the Luminex instruments to detect xNA analytes, producing higher signals without the possibility of competition from any natural oligonucleotides, even in complex biological samples.

Labeled Nucleoside Triphosphates with Reversibly Terminating Aminoalkoxyl Groups

Nucleoside triphosphates having a 3′-ONH2 blocking group have been prepared with and without fluorescent tags on their nucleobases. DNA polymerases were identified that accepted these, adding a single nucleotide to the 3′-end of a primer in a template-directed extension reaction that then stops. Nitrite chemistry was developed to cleave the 3′-ONH2 group under mild conditions to allow continued primer extension. Extension-cleavage-extension cycles in solution were demonstrated with untagged nucleotides and mixtures of tagged and untagged nucleotides. Multiple extension-cleavage-extension cycles were demonstrated on an Intelligent Bio-Systems Sequencer, showing the potential of the 3′-ONH2 blocking group in “next generation sequencing.”

Dynamic assembly of primers on nucleic acid templates

A strategy is presented that uses dynamic equlibria to assemble in situ composite DNA polymerase primers, having lengths of 14 or 16 nt, from DNA fragments that are 6 or 8 nt in length. In this implementation, the fragments are transiently joined under conditions of dynamic equilibrium by an imine linker, which has a dissociation constant of ∼1 μM. If a polymerase is able to extend the composite, but not the fragments, it is possible to prime the synthesis of a target DNA molecule under conditions where two useful specificities are combined: (i) single nucleotide discrimination that is characteristic of short oligonucleotide duplexes (four to six nucleobase pairs in length), which effectively excludes single mismatches, and (ii) an overall specificity of priming that is characteristic of long (14 to 16mers) oligonucleotides, potentially unique within a genome. We report here the screening of a series of polymerases that combine an ability not to accept short primer fragments with an ability to accept the long composite primer held together by an unnatural imine linkage. Several polymerases were found that achieve this combination, permitting the implementation of the dynamic combinatorial chemical strategy.

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.

Synthetic Biology for Improved Personalized Medicine

Tools to re-sequence the genomes of individual patients having well described medical histories is the first step required to connect genetic information to diagnosis, prognosis, and treatment. There is little doubt that in the future, genomics will influence the choice of therapies for individual patients based on their specific genetic inheritance, as well as the genetic defects that led to disease. Cost is the principle obstacle preventing the realization of this vision. Unless the interesting parts of a patient genome can be resequenced for less than

Directed Evolution of Polymerases To Accept Nucleotides with Nonstandard Hydrogen Bond Patterns

10,000 (as opposed to

Transcription, reverse transcription, and analysis of RNA containing artificial genetic components.

100,000 or more), it will be difficult to start the discovery process that will enable this vision. While instrumentation and biology are important to reducing costs, the key element to cost-effective personalized genomic sequencing will be new chemical reagents that deliver capabilities that are not available from standard DNA. Scientists at the Foundation for Applied Molecular Evolution and the Westheimer Institute have developed several of these, which will be the topic of this talk..