Sequencing by avidity enables high accuracy with low reagent consumption.

We present avidity sequencing, a sequencing chemistry that separately optimizes the processes of stepping along a DNA template and that of identifying each nucleotide within the template. Nucleotide identification uses multivalent nucleotide ligands on dye-labeled cores to form polymerase-polymer-nucleotide complexes bound to clonal copies of DNA targets. These polymer-nucleotide substrates, termed avidites, decrease the required concentration of reporting nucleotides from micromolar to nanomolar and yield negligible dissociation rates.

New codons for efficient production of unnatural proteins in a semisynthetic organism.

Natural organisms use a four-letter genetic alphabet that makes available 64 triplet codons, of which 61 are sense codons used to encode proteins with the 20 canonical amino acids. We have shown that the unnatural nucleotides dNaM and dTPT3 can pair to form an unnatural base pair (UBP) and allow for the creation of semisynthetic organisms (SSOs) with additional sense codons. Here, we report a systematic analysis of the unnatural codons.

Optimization of Replication, Transcription, and Translation in a Semi-Synthetic Organism.

Previously, we reported the creation of a semi-synthetic organism (SSO) that stores and retrieves increased information by virtue of stably maintaining an unnatural base pair (UBP) in its DNA, transcribing the corresponding unnatural nucleotides into the codons and anticodons of mRNAs and tRNAs, and then using them to produce proteins containing noncanonical amino acids (ncAAs). Here we report a systematic extension of the effort to optimize the SSO by exploring a variety of deoxy- and ribonucleotide analogues. Importantly, this includes the first in vivo structure-activity relationship (SAR) analysis of unnatural ribonucleoside triphosphates.

Eight-Letter DNA.

Steve Benner and collaborators have recently reported an analysis of DNA containing eight nucleotide letters, the four natural letters (dG, dC, dA, and dT) and four additional letters (dP, dZ, dS, and dB). Their analysis demonstrates that the additional letters do not perturb the structure or stability of the base pairs formed between the natural letters and, remarkably, that the new base pairs, dP-dZ and dS-dB, behave virtually identically to the natural base pairs. This unprecedented result convincingly demonstrates that the thermodynamic and structural behavior previously thought to be the purview of only natural DNA is in fact not unique and can be imparted to suitably designed synthetic components.

Progress Toward a Semi-Synthetic Organism with an Unrestricted Expanded Genetic Alphabet.

We have developed a family of unnatural base pairs (UBPs), exemplified by the pair formed between dNaM and dTPT3, for which pairing is mediated not by complementary hydrogen bonding but by hydrophobic and packing forces. These UBPs enabled the creation of the first semisynthetic organisms (SSOs) that store increased genetic information and use it to produce proteins containing noncanonical amino acids. However, retention of the UBPs was poor in some sequence contexts.

Expansion of the genetic code via expansion of the genetic alphabet.

Current methods to expand the genetic code enable site-specific incorporation of non-canonical amino acids (ncAAs) into proteins in eukaryotic and prokaryotic cells. However, current methods are limited by the number of codons possible, their orthogonality, and possibly their effects on protein synthesis and folding. An alternative approach relies on unnatural base pairs to create a virtually unlimited number of genuinely new codons that are efficiently translated and highly orthogonal because they direct ncAA incorporation using forces other than the complementary hydrogen bonds employed by their natural counterparts.

Chemical Stabilization of Unnatural Nucleotide Triphosphates for the in Vivo Expansion of the Genetic Alphabet.

We have developed an unnatural base pair (UBP) and a semisynthetic organism (SSO) that imports the constituent unnatural nucleoside triphosphates and uses them to replicate DNA containing the UBP. However, propagation of the UBP is at least in part limited by the stability of the unnatural triphosphates, which are degraded by cellular and secreted phosphatases. To circumvent this problem, we now report the synthesis and evaluation of unnatural triphosphates with their β,γ-bridging oxygen replaced with a difluoromethylene moiety, yielding dNaMTP and dTPT3TP.

CalFluors: A Universal Motif for Fluorogenic Azide Probes across the Visible Spectrum.

Fluorescent bioorthogonal smart probes across the visible spectrum will enable sensitive visualization of metabolically labeled molecules in biological systems. Here we present a unified design, based on the principle of photoinduced electron transfer, to access a panel of highly fluorogenic azide probes that are activated by conversion to the corresponding triazoles via click chemistry. Termed the CalFluors, these probes possess emission maxima that range from green to far red wavelengths, and enable sensitive biomolecule detection under no-wash conditions.