Structural basis for TNA synthesis by an engineered TNA polymerase.

Darwinian evolution experiments carried out on xeno-nucleic acid (XNA) polymers require engineered polymerases that can faithfully and efficiently copy genetic information back and forth between DNA and XNA. However, current XNA polymerases function with inferior activity relative to their natural counterparts. Here, we report five X-ray crystal structures that illustrate the pathway by which α-(L)-threofuranosyl nucleic acid (TNA) triphosphates are selected and extended in a template-dependent manner using a laboratory-evolved polymerase known as Kod-RI. Structural comparison of the apo, binary, open and closed ternary, and translocated product detail an ensemble of interactions and conformational changes required to promote TNA synthesis. Close inspection of the active site in the closed ternary structure reveals a sub-optimal binding geometry that explains the slow rate of catalysis. This key piece of information, which is missing for all naturally occurring archaeal DNA polymerases, provides a framework for engineering new TNA polymerase variants.

A Gram-Scale HPLC-Free Synthesis of TNA Triphosphates Using an Iterative Phosphorylation Strategy.

α-l-Threofuranosyl nucleoside 3'-triphosphates (tNTPs) bearing the four genetic bases adenine (A), cytosine (C), guanine (G), and thymine (T) were synthesized on a gram scale using an iterative phosphorylation strategy that avoids the need for tedious HPLC purification. This new synthetic procedure greatly increases the scale on which tNTP substrates can be produced for polymerase-mediated TNA synthesis studies.

A one-pot synthesis of α-l-threofuranosyl nucleoside triphosphates (tNTPs).

TNA (α-l-threofuranosyl nucleoside) triphosphates of adenosine (tATP), guanosine (tGTP), cytidine (tCTP), and thymidine (tTTP) were synthesized from their corresponding 3'-O-phosphoramidite derivatives using a novel one-pot reaction that is less moisture sensitive than traditional methods. The chemically synthesized tNTPs, despite containing an unnatural 3'-triphosphate moiety, are similar in thermal stability to natural nucleotide triphosphates.

A general strategy for expanding polymerase function by droplet microfluidics.

Polymerases that synthesize artificial genetic polymers hold great promise for advancing future applications in synthetic biology. However, engineering natural polymerases to replicate unnatural genetic polymers is a challenging problem. Here we present droplet-based optical polymerase sorting (DrOPS) as a general strategy for expanding polymerase function that employs an optical sensor to monitor polymerase activity inside the microenvironment of a uniform synthetic compartment generated by microfluidics. We validated this approach by performing a complete cycle of encapsulation, sorting and recovery on a doped library and observed an enrichment of ∼1,200-fold for a model engineered polymerase. We then applied our method to evolve a manganese-independent α-L-threofuranosyl nucleic acid (TNA) polymerase that functions with >99% template-copying fidelity. Based on our findings, we suggest that DrOPS is a versatile tool that could be used to evolve any polymerase function, where optical detection can be achieved by Watson-Crick base pairing.

Evaluating TNA stability under simulated physiological conditions.

Chemically modified oligonucleotides are routinely used as diagnostic and therapeutic agents due to their enhanced biological stability relative to natural DNA and RNA. Here, we examine the biological stability of α-l-threofuranosyl nucleic acid (TNA), an artificial genetic polymer composed of repeating units of α-l-threofuranosyl sugars linked by 2',3'-phosphodiester bonds. We show that TNA remains undigested after 7days of incubation in the presence of either 50% human serum or human liver microsomes and is stable against snake venom phosphordiesterase (a highly active 3' exonuclease). We further show that TNA will protect internal DNA residues from nuclease digestion and shield complementary RNA strands from RNA degrading enzymes. Together, these results demonstrate that TNA is an RNA analogue with high biological stability.

A Scalable Synthesis of α-L-Threose Nucleic Acid Monomers.

Recent advances in polymerase engineering have made it possible to copy information back and forth between DNA and artificial genetic polymers composed of TNA (α-L-threofuranosyl-(3',2') nucleic acid). This property, coupled with enhanced nuclease stability relative to natural DNA and RNA, warrants further investigation into the structural and functional properties of TNA as an artificial genetic polymer for synthetic biology. Here, we report a highly optimized chemical synthesis protocol for constructing multigram quantities of TNA nucleosides that can be readily converted to nucleoside 2'-phosphoramidites or 3'-triphosphates for solid-phase and polymerase-mediated synthesis, respectively. The synthetic protocol involves 10 chemical transformations with three crystallization steps and a single chromatographic purification, which results in an overall yield of 16-23% depending on the identity of the nucleoside (A, C, G, T).

Automated solid-phase synthesis of high capacity oligo-dT cellulose for affinity purification of poly-A tagged biomolecules.

Affinity purification of poly-adenylated biomolecules using solid supports that are derivatized with poly-thymidine oligonucleotides provides a powerful method for isolating cellular mRNA. These systems have also been used to purify mRNA-peptide fusions generated by RNA-display. However, the commercial source for high capacity oligo-dT cellulose was recently discontinued. To overcome this problem, we have developed a low cost solid-phase synthesis protocol to generate oligo-dT cellulose. Comparative binding studies indicate that chemically synthesized oligo-dT cellulose functions with superior loading capacity when compared to the discontinued product. We suggest that this method could be used to synthesize oligo-dT resin for routine purification of poly-adenylated biomolecules.

General approach for characterizing in vitro selected peptides with protein binding affinity.

In vitro selection technologies are important tools for identifying high affinity peptides to proteins of broad medical and biological interest. However, the technological advances that have made it possible to generate long lists of candidate peptides have far outpaced our ability to characterize the binding properties of individual peptides. Here, we describe a low cost strategy to rapidly synthesize, purify, screen, and characterize peptides for high binding affinity. Peptides are assayed in a 96-well dot blot apparatus using membranes that enable partitioning of bound and unbound peptide-protein complexes. We have validated the binding affinity constants produced by this method using known peptide ligands and applied this process to discover five new peptides with nanomolar affinity to human α-thrombin. Given the need for new analytical tools that can accelerate peptide discovery and characterization, we feel that this approach would be useful to a wide range of technologies that utilize high affinity peptides.

Identification and characterization of second-generation invader locked nucleic acids (LNAs) for mixed-sequence recognition of double-stranded DNA.

The development of synthetic agents that recognize double-stranded DNA (dsDNA) is a long-standing goal that is inspired by the promise for tools that detect, regulate, and modify genes. Progress has been made with triplex-forming oligonucleotides, peptide nucleic acids, and polyamides, but substantial efforts are currently devoted to the development of alternative strategies that overcome the limitations observed with the classic approaches. In 2005, we introduced Invader locked nucleic acids (LNAs), i.e., double-stranded probes that are activated for mixed-sequence recognition of dsDNA through modification with "+1 interstrand zippers" of 2'-N-(pyren-1-yl)methyl-2'-amino-α-l-LNA monomers. Despite promising preliminary results, progress has been slow because of the synthetic complexity of the building blocks. Here we describe a study that led to the identification of two simpler classes of Invader monomers. We compare the thermal denaturation characteristics of double-stranded probes featuring different interstrand zippers of pyrene-functionalized monomers based on 2'-amino-α-l-LNA, 2'-N-methyl-2'-amino-DNA, and RNA scaffolds. Insights from fluorescence spectroscopy, molecular modeling, and NMR spectroscopy are used to elucidate the structural factors that govern probe activation. We demonstrate that probes with +1 zippers of 2'-O-(pyren-1-yl)methyl-RNA or 2'-N-methyl-2'-N-(pyren-1-yl)methyl-2'-amino-DNA monomers recognize DNA hairpins with similar efficiency as original Invader LNAs. Access to synthetically simple monomers will accelerate the use of Invader-mediated dsDNA recognition for applications in molecular biology and nucleic acid diagnostics.

Invaders: Recognition of Double-Stranded DNA by Using Duplexes Modified with Interstrand Zippers of 2'-O-(Pyren-1-yl)methyl-ribonucleotides.

The invasion has begun: Invaders are shown to recognize DNA hairpins in cell-free assays and chromosomal DNA during non-denaturing fluorescence in situ hybridization (nd-FISH) experiments. As Invaders are devoid of inherent sequence limitations, many previously inaccessible DNA targets could become accessible to exogenous control with important ramifications for karyotyping, in vivo imaging, and gene regulation.

Fluorescent intercalator displacement replacement (FIDR) assay: determination of relative thermodynamic and kinetic parameters in triplex formation--a case study using triplex-forming LNAs.

Triplex forming oligonucleotides (TFOs) are the most commonly used approach for site-specific targeting of double stranded DNA (dsDNA). Important parameters describing triplex formation include equilibrium binding constants (K(eq)) and association/dissociation rate constants (k(on) and k(off)). The 'fluorescent intercalator displacement replacement' (FIDR) assay is introduced herein as an operationally simple approach toward determination of these parameters for triplexes involving TC-motif TFOs. Briefly described, relative rate constants are determined from fluorescence intensity changes upon: (i) TFO-mediated displacement of pre-intercalated and fluorescent ethidium from dsDNA targets (triplex association) and (ii) Watson-Crick complement-mediated displacement of the TFO and replacement with ethidium (triplex dissociation). The assay is used to characterize triplexes between purine-rich dsDNA targets and TC-motif TFOs modified with six different locked nucleic acid (LNA) monomers, i.e. conventional and C5-alkynyl-functionalized LNA and α-L-LNA pyrimidine monomers. All of the studied monomers increase triplex stability by decreasing the triplex dissociation rate. LNA-modified TFOs form more stable triplexes than α-L-LNA-modified counterparts owing to slower triplex dissociation. Triplexes modified with C5-(3-aminopropyn-1-yl)-LNA-U monomer Z are particularly stable. The study demonstrates that three affinity-enhancing features can be combined into one high-affinity TFO monomer: conformational restriction of the sugar ring, expansion of the pyrimidine π-stacking surface and introduction of an exocyclic amine.

C2'-pyrene-functionalized triazole-linked DNA: universal DNA/RNA hybridization probes.

Development of universal hybridization probes, that is, oligonucleotides displaying identical affinity toward matched and mismatched DNA/RNA targets, has been a longstanding goal due to potential applications as degenerate PCR primers and microarray probes. The classic approach toward this end has been the use of "universal bases" that either are based on hydrogen-bonding purine derivatives or aromatic base analogues without hydrogen-bonding capabilities. However, development of probes that result in truly universal hybridization without compromising duplex thermostability has proven challenging. Here we have used the "click reaction" to synthesize four C2'-pyrene-functionalized triazole-linked 2'-deoxyuridine phosphoramidites. We demonstrate that oligodeoxyribonucleotides modified with the corresponding monomers display (a) minimally decreased thermal affinity toward DNA/RNA complements relative to reference strands, (b) highly robust universal hybridization characteristics (average differences in thermal denaturation temperatures of matched vs mismatched duplexes involving monomer W are <1.7 °C), and (c) exceptional affinity toward DNA targets containing abasic sites opposite of the modification site (ΔT(m) up to +25 °C). The latter observation, along with results from absorption and fluorescence spectroscopy, suggests that the pyrene moiety is intercalating into the duplex whereby the opposing nucleotide is pushed into an extrahelical position. These properties render C2'-pyrene-functionalized triazole-linked DNA as promising universal hybridization probes for applications in nucleic acid chemistry and biotechnology.

Invader LNA: efficient targeting of short double stranded DNA.

Despite progress with triplex-forming oligonucleotides or helix-invading peptide nucleic acids (PNAs), there remains a need for probes facilitating sequence-unrestricted targeting of double stranded DNA (dsDNA) at physiologically relevant conditions. Invader LNA probes, i.e., DNA duplexes with "+1 interstrand zipper arrangements" of intercalator-functionalized 2'-amino-alpha-l-LNA monomers, are demonstrated herein to recognize short mixed sequence dsDNA targets. This approach, like pseudo-complementary PNA (pcPNA), relies on relative differences in stability between probe duplexes and the corresponding probe:target duplexes for generation of a favourable thermodynamic gradient. Unlike pcPNA, Invader LNA probes take advantage of the "nearest neighbour exclusion principle", i.e., intercalating units of Invader LNA monomers are poorly accommodated in probe duplexes but extraordinarily well tolerated in probe-target duplexes (DeltaT(m)/modification up to +11.5 degrees C). Recognition of isosequential dsDNA-targets occurs: a) at experimental temperatures much lower than the thermal denaturation temperatures (T(m)'s) of Invader LNAs or dsDNA-targets, b) at a wide range of ionic strengths, and c) with good mismatch discrimination. Recognition of dsDNA is monitored in real-time using inherent pyrene-pyrene excimer signals of Invader LNA probes, which provides insights into reaction kinetics and enables rational design of probes. These properties render Invader LNAs as promising probes for biomedical applications entailing sequence-unrestricted recognition of dsDNA.

Optimized DNA-targeting using triplex forming C5-alkynyl functionalized LNA.

Triplex forming oligonucleotides (TFOs) modified with C5-alkynyl functionalized LNA (locked nucleic acid) monomers display extraordinary thermal affinity toward double stranded DNA targets, excellent discrimination of Hoogsteen-mismatched targets, and high stability against 3?-exonucleases.

Visualization of enantiomers in the liquid-crystalline phase of a fragmented DNA solution.

The use of the liquid-crystalline phase of fragmented DNA solution for enantiomeric differentiation by NMR is reported. The lyotropic cholesteric liquid crystal system formed, orients in a magnetic field and is able to discriminate water soluble enantiomeric mixtures in a simple 2D J-resolved NMR experiment.

Functionalized 2'-amino-alpha-L-LNA: directed positioning of intercalators for DNA targeting.

Chemically modified oligonucleotides are increasingly applied in nucleic acid based therapeutics and diagnostics. LNA (locked nucleic acid) and its diastereomer alpha-L-LNA are two promising examples thereof that exhibit increased thermal and enzymatic stability. Herein, the synthesis, biophysical characterization, and molecular modeling of N2'-functionalized 2'-amino-alpha-L-LNA is described. Chemoselective N2'-functionalization of protected amino alcohol 1 followed by phosphitylation afforded a structurally varied set of target phosphoramidites, which were incorporated into oligodeoxyribonucleotides. Incorporation of pyrene-functionalized building blocks such as 2'-N-(pyren-1-yl)carbonyl-2'-amino-alpha-L-LNA (monomer X) led to extraordinary increases in thermal affinity of up to +19.5 degrees C per modification against DNA targets in particular. In contrast, incorporation of building blocks with small nonaromatic N2'-functionalities such as 2'-N-acetyl-2'-amino-alpha-L-LNA (monomer V) had detrimental effects on thermal affinity toward DNA/RNA complements with decreases of as much as -16.5 degrees C per modification. Extensive thermal DNA selectivity, favorable entropic contributions upon duplex formation, hybridization-induced bathochromic shifts of pyrene absorption maxima and increases in circular dichroism signal intensity, and molecular modeling studies suggest that pyrene-functionalized 2'-amino-alpha-L-LNA monomers W-Y having short linkers between the bicyclic skeleton and the pyrene moiety allow high-affinity hybridization with DNA complements and precise positioning of intercalators in nucleic acid duplexes. This rigorous positional control has been utilized for the development of probes for emerging therapeutic and diagnostic applications focusing on DNA targeting.