Mikhail Abramov

Synthetic genetic polymers capable of heredity and evolution.

Unnatural Bases The genetic basis of all life on the planet is comprised of deoxyribonucleic acid (DNA) with four nitrogenous nucleotide bases, abbreviated to A, G, C, and T. But there are variations on this theme, and Pinheiro et al. (p. 341; see the Perspective by Joyce) describe the directed evolution of unnatural nucleic acid–like genetic polymers. Variant enzymes were developed that efficiently transcribed DNA to anhydrohexitol (HNA), cyclohexenyl (CeNA), locked (LNA), and threofuranosyl (TNA) nuceic acid analogs. Further variant enzymes were developed to reverse-transcribe these analogs back to DNA. Thus, man-made nucleic acid analogs can be designed and selected that have the potential to operate in a way analogous to the natural process of heredity and evolution. Artificial polymers of nucleic acid–like subunits not found in nature can mimic the functions of DNA and RNA. Genetic information storage and processing rely on just two polymers, DNA and RNA, yet whether their role reflects evolutionary history or fundamental functional constraints is currently unknown. With the use of polymerase evolution and design, we show that genetic information can be stored in and recovered from six alternative genetic polymers based on simple nucleic acid architectures not found in nature [xeno-nucleic acids (XNAs)]. We also select XNA aptamers, which bind their targets with high affinity and specificity, demonstrating that beyond heredity, specific XNAs have the capacity for Darwinian evolution and folding into defined structures. Thus, heredity and evolution, two hallmarks of life, are not limited to DNA and RNA but are likely to be emergent properties of polymers capable of information storage.

A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity

The use of chemically synthesized short interfering RNAs (siRNAs) is currently the method of choice to manipulate gene expression in mammalian cell culture, yet improvements of siRNA design is expectably required for successful application in vivo. Several studies have aimed at improving siRNA performance through the introduction of chemical modifications but a direct comparison of these results is difficult. We have directly compared the effect of 21 types of chemical modifications on siRNA activity and toxicity in a total of 2160 siRNA duplexes. We demonstrate that siRNA activity is primarily enhanced by favouring the incorporation of the intended antisense strand during RNA-induced silencing complex (RISC) loading by modulation of siRNA thermodynamic asymmetry and engineering of siRNA 3′-overhangs. Collectively, our results provide unique insights into the tolerance for chemical modifications and provide a simple guide to successful chemical modification of siRNAs with improved activity, stability and low toxicity.

Inhibition of MDR1 expression with altritol-modified siRNAs

Altritol-modified nucleic acids (ANAs) support RNA-like A-form structures when included in oligonucleotide duplexes. Thus altritol residues seem suitable as candidates for the chemical modification of siRNAs. Here we report that ANA-modified siRNAs targeting the MDR1 gene can exhibit improved efficacy as compared to unmodified controls. This was particularly true of ANA modifications at or near the 3′ end of the sense or antisense strands, while modification at the 5′ end of the antisense strand resulted in complete loss of activity. Multiple ANA modifications within the sense strand were also well tolerated. Duplexes with ANA modifications at appropriate positions in both strands were generally more effective than duplexes with one modified and one unmodified strand. Initial evidence suggests that the loss of activity associated with ANA modification of the 5′-antisense strand may be due to reduced phosphorylation at this site by cellular kinases. Treatment of drug resistant cells with MDR1-targeted siRNAs resulted in reduction of P-glycoprotein (Pgp) expression, parallel reduction in MDR1 message levels, increased accumulation of the Pgp substrate rhodamine 123, and reduced resistance to anti-tumor drugs. Interestingly, the duration of action of some of the ANA-modified siRNAs was substantially greater than that of unmodified controls. These observations suggest that altritol modifications may be helpful in developing siRNAs with enhanced pharmacological effectiveness.

HNA and ANA high-affinity arrays for detections of DNA and RNA single-base mismatches.

DNA microarrays and sensors have become essential tools in the functional analysis of sequence information. Recently we reported that chimeric hexitol (HNA) and altritol (ANA) nucleotide monomers with an anhydrohexitol sugar moiety are easily available and proved their chemistry to be compatible with DNA and RNA synthesis. In this communication we describe a novel analytical platform based on HNA and ANA units to be used as synthetic oligonucleotide arrays on a glass solid support for match/mismatch detection of DNA and RNA targets. Arrays were fabricated by immobilization of diene-modified oligonucleotides on maleimido-activated glass slides. To demonstrate the selectivity and sensitivity of the HNA/ANA arrays and to compare their properties with regular DNA arrays, sequences in the reverse transcriptase gene (codon 74) and the protease gene of HIV-1 (codon 10) were selected. Both, the relative intensity of the signal and match/mismatch discrimination increased up to fivefold for DNA targets and up to 3-3.5-fold for RNA targets applying HNA or ANA arrays (ANA>HNA>DNA). Certainly in the new field of miRNA detection, ANA arrays could prove very beneficial and their properties should be investigated in more detail.

Binary Genetic Cassettes for Selecting XNA-Templated DNA Synthesis In Vivo†

Information transfer between natural nucleic acids (DNA and RNA) and xenobiotic nucleic acids (XNA) is rapidly gaining momentum for extending the range of chemical constitutions and the format of molecular evolution accessible to living organisms. Artificial coding by nucleic acid analogues previously focused on structural alterations of base pairs to expand the alphabet of genetic messages. Studies were mostly conducted ex vivo and few experiments have succeeded in vivo thus far. Kool and collaborators demonstrated that size-expanded nucleobases can serve as template for DNA synthesis in E. coli. Substitution of thymine for 5chlorouracil in a whole genome could be performed through automated evolution of E. coli. Conveying genetic information to DNA from an XNA with a chemically deviant backbone is amenable to tight metabolic selection, as demonstrated for hexitol nucleic acid (HNA) using the thymidylate synthase screen in E. coli. We have now shown that various combinations of only the two bases guanine and thymine can be used to encode the active site of thymidylate synthase. This finding was exploited to simplify the synthesis of XNA to be assayed as templates for DNA biosynthesis in vivo, by halving the alphabet needed for this purpose. It could thus be demonstrated that cyclohexenyl nucleic acid (CeNA) can serve in vivo as template, mobilizing a limited effort of chemical synthesis. Further simplification of the binary system to uracil and hypoxanthine enabled to reprogram E. coli with templates simultaneously bearing noncanonical bases and a noncanonical backbone, namely arabinofuranosyl nucleic acid (AraNA) and HNA. A functional thyA gene encoding thymidylate synthase is absolutely required by E. coli cells to grow in nutrient medium devoid of thymine or thymidine (TLM, thymidineless medium). We took advantage of this selection scheme for constructing a plasmid carrying a defective thyA gene in which the six codons specifying the active site around the cysteine at position 146 have been deleted, leaving a gap when digested with the restriction enzymes NheI and NsiI. Mosaic DNA oligonucleotides in which several of the six codons are carried by an XNA backbone can be tested for informational transfer simply by selecting for active thyA genes after transformation of the thyA-deficient strain G929 with heteroduplex ligation products (Figure 1). Up to six contiguous HNA nucleotides were found to serve as a short template for E. coli replication enzymes.

Solution Structure and Conformational Dynamics of Deoxyxylonucleic Acids (dXNA): An Orthogonal Nucleic Acid Candidate

Orthogonal nucleic acids are chemically modified nucleic acid polymers that are unable to transfer information with natural nucleic acids and thus can be used in synthetic biology to store and transfer genetic information independently. Recently, it was proposed that xylose-DNA (dXNA) can be considered to be a potential candidate for an orthogonal system. Herein, we present the structure in solution and conformational analysis of two self-complementary, fully modified dXNA oligonucleotides, as determined by CD and NMR spectroscopy. These studies are the initial experimental proof of the structural orthogonality of dXNAs. In aqueous solution, dXNA duplexes predominantly form a linear ladderlike (type-1) structure. This is the first example of a furanose nucleic acid that adopts a ladderlike structure. In the presence of salt, an equilibrium exists between two types of duplex form. The corresponding nucleoside triphosphates (dXNTPs) were synthesized and evaluated for their ability to be incorporated into a growing DNA chain by using several natural and mutant DNA polymerases. Despite the structural orthogonality of dXNA, DNA polymerase β mutant is able to incorporate the dXNTPs, showing DNA-dependent dXNA polymerase activity.

Synthesis and biological evaluation of inosine phosphonates

The 4′-phosphonomethoxy analogs of inosine and 2′,3′-dideoxyinosine were synthesized and tested for their activity against HCV and HIV, but found to be inactive. During the course of this synthetic investigation, an unexpected oxidative cleavage of the 2′,3′-bond of 2′-deoxyinosine was observed.

Synthesis of Altritol Nucleoside Phosphoramidites for Oligonucleotide Synthesis

This unit describes in detail the optimized preparations of altritol nucleoside phosphoramidite building blocks for oligonucleotide synthesis (aA, aG, aC, aU). D‐Altritol nucleosides with adenine and uracil bases are obtained by nucleophilic opening of the epoxide ring of 1,5:2,3‐dianhydro‐4,6‐O‐benzylidene‐D‐allitol using the 1,8‐diazabicyclo[5.4.0]undec‐7‐ene salts of the above‐mentioned salts, while phase transfer catalysis (18‐crown‐6, K2CO3) is optimal for alkylation of 2‐amino‐6‐chloropurine. The cytosine nucleoside is synthesized starting from the uracil congener. The 3′‐hydroxyl function of hexitol sugar is protected with the benzoyl group. Curr. Protoc. Nucleic Acid Chem. 30:1.18.1‐1.18.21.

Innate potential of random genetic oligomer pools for recombination

The spontaneous emergence of function from prebiotic pools of informational polymers is a central conjecture of current origin of life scenarios. However, the innate functional capacity of random genetic polymer pools is unknown. Here, we have examined the ab initio activity of random and semi-random eicosamer pools of RNA, DNA and the unnatural genetic polymers ANA (arabino-), HNA (hexitol-) and AtNA (altritol-nucleic acids) with respect to a simple functional test: the capacity for intermolecular ligation and recombination. While DNA, ANA and HNA pools proved inert, naïve RNA and AtNA pools displayed diverse modes of intermolecular recombination in eutectic ice phases. Recombination appears linked to the vicinal ring cis-diol shared by RNA and AtNA. Thus, the chemical configuration that renders both susceptible to hydrolysis also enables substantial spontaneous intrapool recombination in the absence of activation chemistry with a concomitant increase in the compositional and structural complexity of recombined pools.

Crystallization and preliminary X-ray study of the d-altritol oligonucleotide GTGTACAC

In altritol nucleic acids (ANAs), the natural five-membered ribose ring of RNA is replaced by the six-membered D-altritol ring. ANAs are good candidates to act as siRNAs in the RNA-interference pathway. Crystals of the fully modified altritol self-complementary octamer GTGTACAC were grown by the hanging-drop vapour-diffusion technique at 289 K. Diffraction data were recorded on SLS beamline X06DA and processed to 3.0 A resolution. The crystals belonged to the hexagonal space group P6(1)22 or P6(5)22, with unit-cell parameters a = 25.05, c = 117.58 A.