Although it is technically complex, atriobiventricular pacing significantly improves exercise tolerance and quality of life in patients with chronic heart failure and intraventricular conduction delay.

Among patients with a history of idiopathic ventricular fibrillation, there is an increased prevalence of early repolarization.

Multiple PV foci are involved in initiation of AF, and elimination of PV muscle conduction is associated with clinical success.

No abstract

Organisms are defined by the information encoded in their genomes, and since the evolution of life, this information has been encoded using a two base pair genetic alphabet (A-T and G-C). In vitro, the alphabet has been expanded to include several unnatural base pairs (UBPs)1–3. We have developed a class of UBPs formed between nucleotides bearing hydrophobic nucleobases, exemplified by the pair formed between d5SICS and dNaM (d5SICS-dNaM, Fig. 1a), which is efficiently PCR amplified1 and transcribed4,5 in vitro, and whose unique mechanism of replication has been characterized6,7. However, expansion of a organism’s genetic alphabet presents new and unprecedented challenges: the unnatural nucleoside triphosphates must enter the cell; endogenous polymerases must be able to faithfully incorporate the unnatural triphosphates into DNA within the complex cellular milieu; and finally, the UBP must be stable in the presence of pathways that maintain the integrity of DNA. Here we show that an exogenously expressed algal nucleotide triphosphate transporter efficiently imports the triphosphates of both d5SICS and dNaM (d5SICSTP and dNaMTP) into E. coli, and that the endogenous replication machinery uses them to accurately replicate a plasmid containing d5SICS-dNaM. Neither the presence of the unnatural triphosphates nor the replication of the UBP introduces a significant growth burden. Lastly, we find that the UBP is not efficiently excised by DNA repair pathways. Thus, the resulting bacterium is the first organism to stably propagate an expanded genetic alphabet.

The clinical benefits of BiV pacing appeared to be significantly maintained over a 12-month follow-up period.

All natural organisms store genetic information in a four-letter, twobase-pair genetic alphabet. The expansion of the genetic alphabet with two synthetic unnatural nucleotides that selectively pair to form an unnatural base pair (UBP) would increase the information storage potential of DNA, and semisynthetic organisms (SSOs) that stably harbor this expanded alphabet would thereby have the potential to store and retrieve increased information. Toward this goal, we previously reported that Escherichia coli grown in the presence of the unnatural nucleoside triphosphates dNaMTP and d5SICSTP, and provided with the means to import them via expression of a plasmid-borne nucleoside triphosphate transporter, replicates DNA containing a single dNaM-d5SICS UBP. Although this represented an important proof-of-concept, the nascent SSO grew poorly and, more problematically, required growth under controlled conditions and even then was unable to indefinitely store the unnatural information, which is clearly a prerequisite for true semisynthetic life. Here, to fortify and vivify the nascent SSO, we engineered the transporter, used a more chemically optimized UBP, and harnessed the power of the bacterial immune response by using Cas9 to eliminate DNA that had lost the UBP. The optimized SSO grows robustly, constitutively imports the unnatural triphosphates, and is able to indefinitely retain multiple UBPs in virtually any sequence context. This SSO is thus a form of life that can stably store genetic information using a six-letter, three-base-pair alphabet.T he natural genetic alphabet is composed of four letters whose selective pairing to form two base pairs underlies the storage and retrieval of virtually all biological information. This alphabet is essentially conserved throughout nature, and has been since the last common ancestor of all life on Earth. Significant effort has been directed toward the development of an unnatural base pair (UBP), formed between two synthetic nucleotides, that functions alongside its natural counterparts (1-3), which would represent a remarkable integration of a man-made, synthetic component into one of life's most central processes. Moreover, semisynthetic organisms (SSOs) that stably harbor such a UBP in their DNA could store and potentially retrieve the increased information, and thereby lay the foundation for achieving the central goal of synthetic biology: the creation of new life forms and functions (4).For over 15 years, we have sought to develop such a UBP (1), and these efforts eventually yielded a family of predominantly hydrophobic UBPs, with that formed between dNaM and d5SICS (dNaM-d5SICS; Fig. 1A) being a particularly promising example (5-7). Despite lacking complementary hydrogen bonding, we demonstrated that the dNaM-d5SICS UBP is well replicated by a variety of DNA polymerases in vitro (7-10), and that this efficient replication is mediated by a unique mechanism that draws upon interbase hydrophobic and packing interactions (11,12). These efforts then culminated in the first pro...

Many candidate unnatural DNA base pairs have been developed, but surprisingly, some of the best replicated adopt intercalated structures in free DNA that are difficult to reconcile with known mechanisms of polymerase recognition. Here we present crystal structures of KlenTaq DNA polymerase at different stages of replicating one of the more promising pairs, dNaM-d5SICS, and show that efficient replication results from the polymerase itself inducing the required natural-like structure.