Towards an Understanding of the Molecular Mechanisms of Variable Unnatural Base-Pair Behavior: A Biophysical Analysis of dNaM-dTPT3.

The series of unnatural base pairs (UBPs) developed by the Romesberg lab, which pair via hydrophobic and packing interactions have been replicated, transcribed, and translated inside of a living organism. However, as to why these UBPs exhibit variable fidelity and efficiency when used in different contexts is not clear. In an effort to gain some insights, we investigated the thermal stability and pairing selectivity of the (d)NaM-(d)TPT3 UBP in 11nt duplexes via UV spectroscopy and the effects on helical structure via CD spectroscopy.

Transcriptional processing of an unnatural base pair by eukaryotic RNA polymerase II.

The development of unnatural base pairs (UBPs) has greatly increased the information storage capacity of DNA, allowing for transcription of unnatural RNA by the heterologously expressed T7 RNA polymerase (RNAP) in Escherichia coli. However, little is known about how UBPs are transcribed by cellular RNA polymerases. Here, we investigated how synthetic unnatural nucleotides, NaM and TPT3, are recognized by eukaryotic RNA polymerase II (Pol II) and found that Pol II is able to selectively recognize UBPs with high fidelity when dTPT3 is in the template strand and rNaMTP acts as the nucleotide substrate.

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.

Nanopore Sequencing of an Expanded Genetic Alphabet Reveals High-Fidelity Replication of a Predominantly Hydrophobic Unnatural Base Pair.

Unnatural base pairs (UBPs) have been developed and used for a variety of applications as well as for the engineering of semisynthetic organisms (SSOs) that store and retrieve increased information. However, these applications are limited by the availability of methods to rapidly and accurately determine the sequence of unnatural DNA. Here we report the development and application of the MspA nanopore to sequence DNA containing the d-d UBP.

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.

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.

The owl sensor: a 'fragile' DNA nanostructure for the analysis of single nucleotide variations.

Analysis of single nucleotide variations (SNVs) in DNA and RNA sequences is instrumental in healthcare for the detection of genetic and infectious diseases and drug-resistant pathogens. Here we took advantage of the developments in DNA nanotechnology to design a hybridization sensor, named the 'owl sensor', which produces a fluorescence signal only when it complexes with fully complementary DNA or RNA analytes. The novelty of the owl sensor operation is that the selectivity of analyte recognition is, at least in part, determined by the structural rigidity and stability of the entire DNA nanostructure rather than exclusively by the stability of the analyte-probe duplex, as is the case for conventional hybridization probes.

Reprograming the Replisome of a Semisynthetic Organism for the Expansion of the Genetic Alphabet.

Semisynthetic organisms (SSOs) created from Escherichia coli can replicate a plasmid containing an unnatural base pair (UBP) formed between the synthetic nucleosides dNaM and dTPT3 (dNaM-dTPT3) when the corresponding unnatural triphosphates are imported via expression of a nucleoside triphosphate transporter. The UBP can also be transcribed and used to translate proteins containing unnatural amino acids. However, UBPs are not well retained in all sequences, limiting the information that can be encoded, and are invariably lost upon extended growth.